# Patent

## Title
Method for detecting target plant genus

## Abstract

A method for detecting species in a target plant genus comprises the steps of conducting PCR using at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a common nucleotide sequence for all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3′ end of primer (A) can complementarily bind to a base in ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3′ end of primer (B) can complementarily bind to a base in ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, and identifying the presence of the resulting amplification product from PCR containing at least a part of ITS-1 or ITS-2 sequence of the target plant genus
The method for detecting species in a target plant genus, particularly an allergenic plant genus such as the genus Fagopyrum, can make it possible to detect with high sensitivity, for example, about 1 ppm of the plant(s) in cases where the plant(s) is contained in a food ingredient or food product.

## Background
CROSS-REFFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 11/581,872, filed Oct. 17, 2006, which is a divisional application of U.S. patent application Ser. No. 10/285,061, filed Oct. 31, 2002, now U.S. Pat. No. 7,144,702, which claims priority to Japanese Patent Application No. 2002-284222 filed on Sep. 27, 2002 and Japanese Patent Application No. 2001-336571, filed Nov. 1, 2001.


BACKGROUND OF THE INVENTION
The 45S rRNA precursor gene sequence (Small Subunit ribosomal RNA (SSU rRNA) gene-Internal Transcribed Spacer-1 (ITS-1) ̃5.8S ribosomal RNA (5.8S rRNA) gene ̃Internal Transcribed Spacer-2 (ITS-2) ̃Large Subunit ribosomal RNA (LSU rRNA) gene) has been used for the classification of species. For example, according to the method developed by Shin J H, et al. (J. Clin. Microbiol., 37: 165-170 (1999)), 5 candida species (fungi) of the genus Candida can be detected and identified using two primers hybridized to the 5.8S rRNA and 28S rRNA (LSU rRNA) gene sequences common to fungi and 5 separate probes each of which can specifically hybridize to the ITS-2 sequence of its corresponding species. The method is different from the present invention as described below. Firstly, the method is aimed at fungi, specifically candida (fungi). Secondly, the method does not use the primers, which hybridize to ITS-1 or ITS-2 sequence. Consequently, these primer pairs do not assure the specificity to the genus Candida, whereas each of five probes can independently recognize its corresponding candida species (fungi) of the genus Candida. In other words, only one species of the genus Candida can be detected and identified when a single set of the primer pair and a probe is used. Thirdly, the above publication does not describe about the sensitivity of the detection, which is very important for the detection methods of allergenic plants in food. Lastly, the method needs expensive reagents and instruments due to the use of probes.
According to the method developed by Proft J, et al. (Parasitol. Res., 85: 837-843 (1999)), a certain anopheles mosquito can be classified into one of 6 species of the genus Anopheles using 6 primer pairs. The method uses a primer that can hybridize to the 5.8 rRNA gene sequence common to the six anopheles mosquito species of the genus Anopheles in combination with 6 primer pairs each of which can specifically hybridize to the ITS-2 sequence of its corresponding anopheles mosquito species of the genus Anopheles. Based on the size of the amplification product obtained by PCR method, the anopheles mosquito of interest can be classified into one of the 6 species of the genus Anopheles. The method is different from the present invention as described below. Firstly, the method is aimed at mosquitoes, specifically the anopheles mosquitoes. Secondly, due to the properties of designed primer pairs, only one species of the genus Anopheles can be detectable when a single primer pair is used. Thirdly, an object of the method is to identify a specimen exclusively derived from a single species of mosquitoes. Consequently, the object of the method is not to analyze anopheles mosquitoes in a mixture. Lastly, the above publication does not describe about the sensitivity of the detection, which is very important for the detection methods of allergenic plants in food.
Thus, the conventional methods mentioned above are to detect one specific species in a mixture and to identify a bio specimen exclusively derived from a single species of the genus, and therefore, the methods do not relate to a method for detecting the target genus broadly in cases where even one kind of the target genus is contained in a mixture. In addition, the primer sequences common to several species are located on SSU rRNA, 5.8S rRNA and LSU rRNA gene sequence, and therefore, primer sequences common to several species are not found in ITS-1 or ITS-2 sequence.
On the other hand, regarding detection of allergenic plants in food, a method for detecting whether some wheat is contained in a food sample of interest is disclosed by Allmann M, et al. (Z Lebensm Unters Forsch, 196: 248-251 (1993)). The method uses primers which specifically hybridize to a IGS sequence between 25S rRNA (LSU rRNA) and 18S rRNA (SSU rRNA) gene sequences of wheat. However, it is hard to evaluate the specificity of the primers by simulation and the like because the primers have to be designed based on little information about the IGS sequence in the method. Therefore, it would be difficult to judge the reliability of the analysis.
SUMMARY OF THE INVENTION
An object of the present inventions is to provide a method for detecting species (a plant or plants) in a target plant genus, particularly an allergenic plant genus such as the genus Fagopyrum, which makes it possible to detect with high sensitivity, for example, about 1 ppm of the plant(s) in cases where the plant(s) is contained in a food ingredient or food product.
Since a trace of allergenic food ingredients, particularly plants in an allergenic plant genus may be unintentionally contaminated in the food ingredient or product at the stages of production, distribution and fabrication, it is important that providers of the food ingredient or product conduct quality control to detect whether these plants have contaminated the food ingredient or product.
For example, regarding buckwheat, though it is reported that patients are affected with anaphylaxis by pillows made of buckwheat chaff and die due to anaphylactic shock and traces of buckwheat may effect a severe symptom in allergic patients for buckwheat, there is no method for detecting buckwheat in the food ingredient or product in the world. For example, it is considered that contamination of buckwheat into the food ingredient or product occurs in a case where buckwheat grown near a field cultivated with plants other than buckwheat is contaminated in the food ingredient harvest time. Therefore, in order to find the contamination of trace of unintended buckwheat, it is desirable that a method for detecting buckwheat be built up, wherein the method can detect as sensitive as possible, for example, even 1 ppm of the buckwheat in a food ingredient and product. Furthermore, as for grain allergies, it is said that some cross-reaction occurs among taxonomically related plants, and therefore, it is desirable that the method be able to detect a wide range of any plants in the genus Fagopyrum without limiting the detectable plants to eatable buckwheat.
Regarding a method for detecting peanuts, an ELISA kit, which can detect about 2.5 ppm of peanuts using specific antibodies for proteins inherent to peanuts, have been sold and used in the world. When positive finding in ELISA, whether it is false positive or truly positive can be confirmed in detail by Western Blot etc., but it confirms only the size of protein involving antigen-antibody reaction. A method for detecting a DNA inherent to peanuts has not been reported. In order to detect peanuts in a food ingredient and product through a variety of processing steps, it is desirable that there is built up a method for detecting target DNA sequences, which will have a relatively high resistivity against the processing rather than proteins Furthermore, as it is the same as in buckwheat, it is desirable that the method be able to detect a wide range of plants in the genus Arachis. 
Thus, it is important to detect a plant(s) in the allergenic plant genus with high sensitivity in cases where even only one kind of the plants is contained in the food ingredient, product and the like.
In cases of genetically modified products and the like, DNA sequences to be detected are limited to recombinant DNA sequences. On the other hand, in cases of plants which originally exist in nature, there has not been clear knowledge how to choose a target DNA sequence from a large number of DNA sequences, and whether the thus chosen DNA sequence is useful and universal for a variety of plants. It has been conducted to choose a specific protein to a target plant, and to detect a DNA sequence coding for the protein, but it is necessary to choose a separate specific protein to each plant. Furthermore, even if such a specific protein can be chosen, when the copy number of a DNA sequence coding for the protein is small, there are some cases where the method may not have a sufficient sensitivity and therefore it will be disadvantage for the detection of traces of a contaminating plant.
Under such circumstances, in order to develop a method for detecting a plant(s) in an allergenic plant genus and the like in cases where even only one kind of the plants is contained in a food ingredient and product, the present inventors have focused their attention on the gene sequences of a target plant genus to vigorously conduct the research. In order to detect whether one specific plant has contaminated a food ingredient or product, it may be conducted to detect a specific gene sequence of the plant in the food ingredient and product. However, in order to detect a case where even only one kind of the plants is contained in a genus in a food ingredient and product, such method is very complicated and inefficient because it is necessary to repeat the same operation for respective plants in a specific genus.
In order to solve this problem, the inventors have conducted further research, collected some information on gene sequences of plants in the genus Fagopyrum (21 sequences registered in GenBank) and in other genus and studied on a variety of viewpoint, and thereby, the inventors have found that a specific common sequence for plants in the genus Fagopyrum, which differs from a sequence of plants in other genus, is present in gene sequences of the plant in the genus Fagopyrum (21 sequences registered in GenBank). As the result of an investigation conducted based on this knowledge for other plant genus such as the genus Arachis, the inventors have also found that there is similar tendency among them.
Based on this knowledge, it has been found that a method for detecting each allergenic plant genus using a sequence of 45S rRNA precursor gene, as a sequence which exists as a sequence having a large copy number in plant DNA and is specific to each allergenic plant genus, can be useful in attaining the object. When positive indication appears in PCR, differently from ELISA, as an amplification product can be analyzed not only in the size thereof but also in detail sequence thereof by sequencing the amplification product, it can be confirmed more precisely whether it is false positive or truly positive. Furthermore, it has been found that, by choosing a region including ITS-1 or ITS-2 sequence as a target sequence, the method is useful in detecting trace of plants in the target plant genus in a mixture because the specific sequence can be obtained and common region of sequences for plants in the genus can be chosen. Moreover, as the sequence of 45S rRNA precursor gene is present in most plants, it can be advantageously applied on a variety of plants.
Based on this knowledge, the present inventions have been completed. In this connection, the following method for detecting plants can be applied not only to the allergenic plant genus but also to other plant genus.
Accordingly, the present invention provides a method for detecting species (a plant(s)) in a target plant genus, which comprises the steps of conducting PCR using at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3′ end of primer (A) can complementarily bind to a base in a ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3′ end of primer (B) can complementarily bind to a base in a ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, and identifying the presence of the resulting amplification product from PCR containing at least a part of the ITS-1 or ITS-2 sequence of the target plant genus.
Herein, the phrase “hybridize under stringent conditions” means that two DNA fragments hybridize to each other under the standard hybridization condition described by Sambrook J. et al. (Expression of Cloned Genes in E. coli (Molecular Cloning: A laboratory Manual (1989)) Cold Spring Harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61). More specifically, for example, it means that a hybridization and washing (for example, about 2.0×SSC, 50° C.) are conducted on the basis of Tm value obtained by the following equation.

Tm=81.5+16.6(log10[Na+])+0.41(fraction G+C)−(600/N)

In addition, the term genus as used in the present specification means a group including all species in the genus or some species chosen from the species in the genus.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.
FIG. 2 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.
FIG. 3 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 1.
FIG. 4 is nucleotide sequences obtained by a sequence analysis of the target amplification product from buckwheat chaff.
FIG. 5 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 3.
FIG. 6 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 3.
FIG. 7 is nucleotide sequences obtained by a sequence analysis of the target amplification product from Shirahana soba.
FIG. 8 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 5.
FIG. 9 is nucleotide sequences obtained by a sequence analysis of the target amplification product from peanut.
FIG. 10 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 6.
FIG. 11 is an electrophoretogram showing the results electrophoresed on a 2% agarose gel in Example 6.
FIG. 12 is nucleotide sequences obtained by a sequence analysis of the target amplification product from peanut.
FIG. 13 is 45S rRNA precursor gene structure.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Although a target plant genus to be detected by the method of the present invention may be any plant genus, because the method can detect a presence of trace of a plant(s) in the target plant genus in a food ingredient or product, the method is particularly useful in detecting whether plants in the allergenic plant genus such as the genus Fagopyrum, genus Arachis, genus Triticum and genus Glycine are contaminated in the food ingredient or product.
The method of the present inventions uses at least one member selected from the group consisting of primers (A) and (S.), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in a 45S rRNA precursor gene sequence thereof, wherein 3′ end of primer (A) can complementarily bind to a base in a ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule, while 3′ end of primer (B) can complementarily bind to a base in a ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule to conduct PCR amplification for DNA isolated from a subject to which the method is to be applied. In the PCR amplification, based on a conventional procedure described in publications, for example, Saiki R K, et al., Science, 230: 1350-1354 (1985) and Shyokubutsu no PCR Zikken Protocol—Idenshi no Tanri—Hatsugen Kara Genome Kaiseki Made—(Saiboukougaku Bessatsu Saiboukougaku Series 2), General Editors Shimamoto, K. and Sasaki, T., Shujunsha Co., Ltd., Tokyo, 1995 and the like, optimal conditions are chosen from appropriate modification of temperature and time of each step of denaturation, annealing and extension, a kind and concentration of enzyme (DNA polymerase), concentrations of dNTP, primer and magnesium chloride, an amount of template DNA and the like.
In addition, PCR amplification may be conducted at an annealing temperature of the primer and the template DNA higher than Tm value of the primer, preferably the Tm value plus 10 to 3° C., and subsequently at an annealing temperature near the Tm value, preferably the Tm value plus 7 to 0° C., wherein the Tm value is determined by commercially available software such as HYB SimulatorTM version 4.0 (Advanced Gene Computing Technologies, Inc.) and Primer ExpressTM version 1.5 (PE Applied Biosystems).
After the PCR amplification of DNA isolated from a subject to be studied such as a food ingredient or product, the resulting reaction solution is analyzed by for example, electrophoresis to determine whether the target plant genus is present in the subject. The determination is based on whether any PCR amplification products having target size are present in the reaction solution after the PCR amplification, and if the PCR amplification products are present in the reaction solution, whether at least a part of the ITS-1 or ITS-2 sequence of the target plant genus is present in the sequence of the PCR amplification products. That is, if the PCR amplification products, which have the target size and contain at least a part of the ITS-1 or ITS-2 sequence of the target plant genus, are present in the reaction solution, the studied subject is contaminated by a plant(s) in the target plant genus. On the other hand, if the PCR amplification products are not present in the reaction solution or even though it exists, unless it contains at least a part of ITS-1 or ITS-2 sequence of the target plant genus, the studied subject is not contaminated by a plant(s) in the target plant genus. Furthermore, the method of the present invention can detect with high sensitivity, for example, about 1 ppm level of a contamination.
For example, at least 2 primers may be used in the method of the present invention. In cases where at least 2 kinds of the target plant genus are detected at the same time, at least 3 primers may be used provided that it is important to use at least one member selected from the group consisting of primers (A) and (B), which can hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence common to all species in the target plant genus in 45S rRNA precursor gene sequence thereof, wherein 3′ end primer (A) can complementarily bind to a base in ITS-1 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule while 3′ end of primer (13) can complementarily bind to a base in ITS-2 sequence of the target plant genus when the primer hybridizes to the nucleic acid molecule. In this connection, examples of the primer (A) include primers, which can hybridize to a nucleic acid molecule having a boundary between a ITS-1 sequence and a 5.8S rRNA gene sequence or which can hybridize to a nucleic acid molecule having a boundary between a ITS-1 sequence and a SSU rRNA gene sequence. Likewise, examples of the primer (1B) include primers, which can hybridize to a nucleic acid molecule having a boundary between a ITS-2 sequence and a 5.8S rRNA gene sequence or which can hybridize to a nucleic acid molecule having a boundary between a ITS-2 sequence and a LSU rRNA gene sequence. Preferably the primers (A) and (B) have at least 15 bases, more preferably 15 to 30 bases. Since the ITS-1 sequence and the ITS-2 sequence contain many specific sequences for species, the primer (A) or (13), which has a specificity common to the target plant genus, can be obtained by choosing a suitable nucleic acid molecule having a specific nucleotide sequence common to the target plant genus in the ITS-1 and ITS-2 sequences, as a nucleic acid molecule having a nucleotide sequence common to the target plant genus in the 45S rRNA precursor gene sequence. One or two or more member(s) selected from the group consisting of the primer (A) and the primer (B) may also be used, but if at least two members are used, the method of the present invention can become more highly sensitive to the target plant genus, particularly genus Fagopyrum. 
In another embodiment of the method for detection of the present invention, primer (A) is used together with a primer (C) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded ITS-1, 5.8S rRNA gene, ITS-2 and LSU rRNA gene of the target plant genus. Alternatively, primer (A) is used together with a primer (E) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded SSU rRNA gene and ITS-1 of the target plant genus. In a further embodiment of the method for detection of the present invention, the primer (B) is used together with a primer (D) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 of the target plant genus. Alternatively, primer (B) is used together with a primer (F) which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence continuously bonded ITS-2 and LSU rRNA gene of the target plant genus. In this connection, 5.8S rRNA gene is highly preservative and contains many sequences common to a great majority of plants. Therefore, as a primer (C), by appropriately choosing a primer, which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence of 5.8S rRNA gene, wherein 3′ end thereof can complementarily bond to a nucleotide sequence in 5.8S rRNA gene sequence when the primer hybridizes to the nucleic acid molecule, or as s primer (D), by appropriately choosing a primer, which can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence of 5.8S rRNA gene, wherein 3′ end thereof can complementarily bond to a nucleotide sequence in 5.8S rRNA gene sequence when the primer hybridizes to the nucleic acid molecule, the resulting primer can be commonly used for a variety of plants. If said primer is fixed and a common specific primer is chosen for the species in the target plant genus from the ITS-1 or ITS-2 region thereof, then the primers can be easily designed to detect with high sensitivity the contaminated plants in the target plant genus. Preferably, the primers (C) to (F) have at least 15 bases, more preferably 15 to 30 bases.
When these primers are designed, it will be sufficient to design them based on, for example, PCR Hou Saizensen—Kisogizyutsu Kara Ouyou Made (Tanpakushitsu•Kakusan•Kouso Rinzizoukan), ed. Sekiya, T. and Fujinaga, K., Kyoritsu Shuppan Co. Ltd., Tokyo, 1997, Baio Zikken Illustrated 3 Hontouni Hueru PCR (Saiboukougaku Besshi Me de Miru Zikken Note Series), Nakayama, H., Shujunsha Co., Ltd., Tokyo, 1996 or PCR Technology: Principles and Applications of DNA Amplification, ed. Erlich, H. A., Stockton Press, Inc., NY, 1989. However, since there is a low possibility that the target DNA is decomposed when the DNA is detected in un-processed materials, the primers may be those which can induce an amplification product within 700 bases, and since there is a possibility that the target DNA is decomposed to become short when the DNA is detected in processed foods, the primers, which can induce an amplification product within 200 bases, are preferable in view of that the primers provide high sensitivity.
In view of the above, it is preferable that the primer (C) or (D) be able to hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence indicated by SEQ NO:1 or a complementary nucleotide sequence thereof. Said primer is preferable because the region indicated by SEQ NO:1 has an especially high homology, a primer which hybridize to any region of 5.8S rRNA gene sequence may be used because the sequences of species in the allergenic plant genus have a high homology over almost the whole region of 5.8S rRNA gene sequence. More preferably, it is a primer, which can hybridize under stringent conditions to a nucleic acid molecule having positions 11 to 63 of the nucleotide sequence of SEQ NO:1 or a complementary nucleotide sequence thereof. Preferably, primer (C) is an oligonucleotide indicated by any of SEQ NO:2, 3 or 4, which hybridizes to the nucleic acid molecule of SEQ NO:1. Preferably, primer (D) is also an oligonucleotide indicated by any of SEQ NO:5, 6 or 7, which hybridizes to a complementary strand of SEQ NO:1. Said primers have to hybridize under stringent conditions specific to the target nucleic acid molecule and 3′ end thereof have to be a complementary base to the target part of DNA sequence so that the hybridized primers can function as one primer and an extension reaction occurs. Therefore, as long as the primers meet the above requirement, the primers may be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:2 to 7, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto.
The specific nucleotide sequence common to the target plant genus in ITS-1 or ITS-2 sequence can be identified by obtaining the ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequence of a plant(s) in the target plant genus to be detected and other plant genus from GenBank, conducting an alignment and searching a region having a high specificity common to the target plant genus. In addition, among the regions thus identified, a base, which can assure that the base is specific to the target plant genus and not to plants thought to be related species thereof, can be determined as 3′ end of the primers to select a primer sequence.
When the target plant genus is the genus Fagopyrum, examples of a commonly specific nucleotide sequences in the ITS-1 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:8, 9 or 10, or a complementary nucleotide sequence thereof. Preferably, they include a nucleotide sequence of positions 11 to 61 of the nucleotide sequence of SEQ NO:8 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 67 of the nucleotide sequence of SEQ NO:9 or a complementary nucleotide sequence thereof. In addition, SEQ NO:10 is particularly useful as a region for selecting primers for detecting specifically F. esculentum (common buckwheat), F. tataricum (Tartarian buckwheat), F. homotropicum and/or F. cymosum, which are members of the genus Fagopyrum. 
Preferably, the primer (A) is an oligonucleotide indicated by any of SEQ NOs:11 to 16 wherein the oligonucleotide indicated by any of SEQ NOs:11 to 14 hybridizes to a complementary strand of SEQ NO:8 and the oligonucleotide indicated by any of SEQ NOs:15 and 16 hybridizes to a nucleic acid molecule of SEQ NO:9. The primer (A) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:11 to 16, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. Furthermore, examples of the common specific nucleotide sequence in ITS-2 include a nucleotide sequence indicated by any of SEQ NO:21 or 22, or a complementary nucleotide sequence thereof. These nucleotide sequences are particularly useful as a region for selecting primers for detecting specifically F. esculentum (common buckwheat), F. tataricum (Tartarian buckwheat), F. homotropicum and/or F. cymosum, which are members of the genus Fagopyrum. In addition, it is preferable to use a combination of the primer of any of SEQ NOs:11 to 14 and the primer of any of SEQ NOs:15, 16 or 2 to 4.
When the target plant genus is the genus Arachis, examples of a common specific nucleotide sequences in the ITS-1 sequence thereof include a nucleotide sequence indicated by SEQ NO:17, or a complementary nucleotide sequence thereof. Preferably, they include a nucleotide sequence of positions 1 to 60 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 43 to 99 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof. More preferably, they include a nucleotide sequence of positions 11 to 50 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 53 to 89 of the nucleotide sequence of SEQ NO:17 or a complementary nucleotide sequence thereof.
Preferably, the primer (A) is an oligonucleotide indicated by any of SEQ NOs:18 to 20, which hybridizes to a complementary strand of SEQ NO:17. The primer (A) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:18 to 20, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. Furthermore, examples of the common specific nucleotide sequence in ITS-2 sequence of the genus Arachis include a nucleotide sequence of SEQ NO:23 or a complementary nucleotide sequence thereof. Preferably, it is a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:23 or a complementary nucleotide sequence thereof. Moreover, it is preferable that the primer (B) be an oligonucleotide indicated by SEQ NO:24, which hybridizes to a nucleic acid molecule of SEQ NO:23. The primer (13) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NO:24, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. In addition, it is preferable to use a combination of the primer of any of SEQ NOs:18 to 20 and the primer of any of SEQ NOs: 2 to 4, a combination of the primer of any of SEQ NOs: 18 to 20 and the primer of SEQ NO:24 or a combination of the primer of SEQ NO:24 and the primer of any of SEQ NOs:5 to 7, and more preferably, a combination of the primer of any of SEQ NOs:18 to 20 and the primer of any of SEQ NOs:2 to 4.
When the target plant genus is genus Triticum, examples of common specific nucleotide sequences in ITS-2 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:25, 26 or 27, or a complementary nucleotide sequence thereof. Preferably, it is a nucleotide sequence of positions 11 to 50 of the nucleotide sequence of SEQ NO:25 or a complementary nucleotide sequence thereof, a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:26 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 47 of the nucleotide sequence of SEQ NO:27 or a complementary nucleotide sequence thereof.
Preferably, the primer (B) is an oligonucleotide indicated by any of SEQ NOs:28 to 30 wherein the oligonucleotide of SEQ NO:28 hybridizes to the complementary strand of SEQ NO:25, the oligonucleotide of SEQ NO:29 hybridizes to the nucleic acid molecule of SEQ NO:26 and the oligonucleotide of SEQ NO:30 hybridizes to the nucleic acid molecule of SEQ NO:27. The primer (B) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:28 to 30, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. In addition, it is preferable to use a combination of the primer of SEQ NO:28 and at least one primer selected from the group consisting of SEQ NOs:29 and 30.
When the target plant genus is genus Glycine, examples of commonly specific nucleotide sequences in ITS-2 sequence thereof include a nucleotide sequence indicated by any of SEQ NO:31, 32 or 33, or a complementary nucleotide sequence thereof. Preferably, it is a nucleotide sequence of positions 11 to 48 of the nucleotide sequence of SEQ NO:31 or a complementary nucleotide sequence thereof, a nucleotide sequence of positions 11 to 55 of the nucleotide sequence of SEQ NO:32 or a complementary nucleotide sequence thereof, or a nucleotide sequence of positions 11 to 52 of the nucleotide sequence of SEQ NO:33 or a complementary nucleotide sequence thereof.
Preferably, the primer (B) is an oligonucleotide indicated by any of SEQ NOs:34 to 41 wherein the oligonucleotide of SEQ NO:34 hybridizes to a complementary strand of SEQ NO:31, the oligonucleotide of any of SEQ NOs:35 to 40 hybridizes to a nucleic acid molecule of SEQ NO:32 and the oligonucleotide of SEQ NO:41 hybridizes to a nucleic acid molecule of SEQ NO:33. The primer (B) may also be an oligonucleotide indicated by any nucleotide sequence of SEQ NOs:34 to 41, wherein one or several base(s) thereof are deleted or substituted, or one or several base(s) are added thereto. It is preferable to use a combination of the primer of SEQ NO:34 and at least one primer selected from the group consisting of SEQ NOs:35 to 41.
In order to design these primers and to evaluate the designed primers, a PCR simulation may be used.
For example, in order to design the primer for detecting the genus Fagopyrum, a common region having a high specificity for all of the 21 DNA sequences of plants in genus Fagopyrum including eatable buckwheat (common buckwheat and Tartarian buckwheat) is selected from the region of ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequence, and further, a base, which can assure the specificity to other plants, is selected as 3′ end of the primer to determine the primer sequence. However, the species in the genus Fagopyrum have the ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequence from which a part thereof is deleted and from which a number of bases are deleted, which differ from each other, and therefore, it is necessary to conduct further selection in order to obtain a same size of amplification product for the 21 plants in the genus Fagopyrum. If the same size of amplification product can be obtained for the 21 plants in the genus Fagopyrum, the presence of the genus Fagopyrum can be easily detected. In the genus Fagopyrum, particularly by selecting the primer (A) and the primer (C) or two primers (A), the simulation has confirmed that the same size of amplification product would be obtained for all of 21 plants in the genus Fagopyrum. There can be designed primers by which nonspecific products can be easily identified in light of the size of the products.
As mentioned above, regarding the designed primer, it was confirmed by PCR simulation whether or not the target amplification product could be obtained and the results were almost the same as the results of actual PCR, and therefore, the simulation results possess high reliability In this connection, the above described PCR simulation software, Amplify 1.0 (Bill Engels) and the like can be used in the PCR simulation.
An amplification of DNA sequence using the primers described above can be conducted by PCR methods (Polymerase Chain Reaction: for example, Saiki R K, et al., Science, 230: 1350-1354 (1985)), as well as LAMP (Loop-Mediated Isothermal Amplification: Notomi T, et al., Nucleic Acids Res., 28 e 63 (2000)) or by other appropriate methods. In addition, though the amplification product is generally detected by electrophoresis, other methods can be used.
EXAMPLES
The present invention will be described more specifically with reference to the following Examples.
Example 1
A. Design of Oligonucleotide Primers for Detection of DNA from Buckwheat
(1) DNA Sequences of the Genus Fagopyrum 
Regarding the genus Fagopyrum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 21 DNA sequences registered in GenBank were examined to select suitable regions for the primers.

    1: Fagopyrum urophyllum (AB000342)
    2: Fagopyrum urophyllum (AB000341)
    3: Tartarian buckwheat: Fagopyrum tataricum (sub_species: potanini) (AB000340)
    4: Tartarian buckwheat: Fagopyrum tataricum (AB000339)
    5: Fagopyrum statice (AB000338)
    6: Fagopyrum statice (AB000337)
    7: Fagopyrum pleioramosum (AB000336)
    8: Fagopyrum lineare (AB000335)
    9: Fagopyrum leptopodum (AB000334)
    10: Fagopyrum homotropicum (AB000333)
    11: Fagopyrum gracilipes (AB000332)
    12: Common buckwheat: Fagopyrum esculentum ancestralis (AB000331)
    13: Common buckwheat: Fagopyrum esculentum (AB000330)
    14: Fagopyrum cymosum (AB000329)
    15: Fagopyrum cymosum (AB000328)
    16: Fagopyrum cymosum (AB000327)
    17: Fagopyrum cymosum (AB000326)
    18: Fagopyrum cymosum (AB000325)
    19: Fagopyrum cymosum (AB000324)
    20: Fagopyrum capillatum (AB000323)
    21: Fagopyrum callianthum (AB000322)

(2) DNA Sequences of Other Common Allergenic Plants



As sequences of peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: peanut: Arachis hypogaea (AF156675)
    2: wheat: Triticum aestivum (AJ301799)
    3: soybean: Glycine max (U60551)
    4: walnut: Juglans regia (AF303809)
    5: matsutake mushroom: 7 Ticholoma matsutake (U62964)
    6: peach: Prunus persica (AF185621)
    7: apple: Malus×domestica (AF186484)
    8: Valencia orange: Citrus sp. (E08821)

(3) DNA Sequences of Plants Widely Used for a Food Ingredient



As sequences of corn, brown rice, pepper and mustard, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: corn: Zea mays (U46648)
    2: brown rice: Oryza sativa (AF169230)
    3: pepper: Piper nigrum (AF275197)
    4: mustard: Sinapis alba (X15915)

(4) Oligonucleotide Primer Synthesis and Evaluation



Among ITS-1 sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum, there was determined a nucleotide sequence which would specifically hybridize to all of the 21 DNA sequences of the genus Fagopyrum through the study of the ITS-1 sequences. The thus determined nucleotide sequence is indicated as SEQ NO:11. Subsequently, the oligonucleotide primer with SEQ NO:11 was synthesized.









Sense primer




5′-GGA CCA CGA ACA GAA GCG CGT CCC G-3′
(SEQ NO:11)






From among 5.8S rRNA gene sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum and 8 DNA sequences of other common allergenic plants, there was determined a nucleotide sequence which would hybridize to all of these sequences through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:3. Subsequently, the oligonucleotide primer with the SEQ NO:3 was synthesized.










Antisense primer









(SEQ NO:3)











5′-ATC GCA TTT CGC TAC GTT CTT CAT CG-3′







Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels). As a result, it was predicted that target 140 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. In contrast, no 140 bp amplification product was predicted from the aforementioned 8 DNA sequences of common allergenic plants other than genus Fagopyrum (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange) and the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard). However, the results of the simulation indicated some possibility that nonspecific amplification products, which were different from the target one in size, would be obtained from soybean, apple and orange in light of weak amplified signals. On the other hand, no amplification product was predicted from the 5 DNA sequences of other common allergenic plants (peanut, wheat, walnut, matsutake mushroom and peach) and the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard). The simulation results are shown in Tables 1A and 1B. The meanings of symbols and numerical values in Tables 1A and 1B are explained below.

    ★: An obtained amplification product whose size almost matched to the target product size 140 bp (+10 bp), which would be obtained.
    W 2-6: Probability of obtaining amplification products
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−) No amplification product was predicted.









TABLE 1A







SEQ NO: 11 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Fagopyrum
★Fagopyrum urophyllum
AB000342
140 bp
—
—
—
—



★Fagopyrum urophyllum
AB000341
140 bp
—
—
—
—



★Fagopyrum tataricum
AB000340
140 bp
—
 64 bp
—
—



(Tartarian buckwheat)



★Fagopyrum tataricum
AB000339
140 bp
—
 64 bp
—
—



(Tartarian buckwheat)



★Fagopyrum statice
AB000338
140 bp
—
—
—
—



★Fagopyrum statice
AB000337
140 bp
—
—
—
—



★Fagopyrum pleioramosum
AB000336
140 bp
—
—
—
—



★Fagopyrum lineare
AB000335
140 bp
—
—
—
—



★Fagopyrum leptopodum
AB000334
140 bp
—
—
—
—



★Fagopyrum homotropicum
AB000333
140 bp
—
326 bp
—
—



★Fagopyrum gracilipes
AB000332
140 bp
—
—
—
—



★Fagopyrum esculentum
AB000331
140 bp
—
326 bp
—
—



(Common buckwheat)



★Fagopyrum esculentum
AB000330
140 bp
—
325 bp
—
—



(Common buckwheat)



★Fagopyrum cymosum
AB000329
140 bp
—
—
333 bp
—



★Fagopyrum cymosum
AB000328
140 bp
—
—
321 bp
—



★Fagopyrum cymosum
AB000327
140 bp
—
—
321 bp
—



★Fagopyrum cymosum
AB000326
140 bp
—
—
—
—



★Fagopyrum cymosum
AB000325
140 bp
—
—
321 bp
—



★Fagopyrum cymosum
AB000324
140 bp
—
—
333 bp
—



★Fagopyrum capillatum
AB000323
140 bp
—
—
—
—



★Fagopyrum callianthum
AB000322
140 bp
—
—
—
—
















TABLE 1B







SEQ NO: 11 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Other Common Allergenic
Arachis hypogaea (Peanut)
AF156675
—
—
—
—
—


Plants
Triticum aestivum (Wheat)
AJ301799
—
—
—
—
—



Glycine max (Soybean)
U60551
—
—
—
227 bp
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
—



Malus x domestica (Apple)
AF186484
—
—
275 bp
—
—



Citrus sp. (Valencia orange)
E08821
—
—
312 bp
—
—







215 bp


Plants Widely Used for a Food
Zea mays (Corn)
U46648
—
—
—
—
—


Ingredient
Oryza sativa (Brown rice)
AF169230
—
—
—
—
—



Piper nigrum (Pepper)
AF275197
—
—
—
—
—



Sinapis alba (Mustard)
X15915
—
—
—
—
—









B. Preparation of Template DNA for PCR

(1) Samples Used for DNA Isolation

Buckwheat:

Commercially available seeds of Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat) were used.
Buckwheat Chaff:
Commercially available buckwheat chaff used for pillows was used.
Pepper:
Commercially available seeds of black pepper and white pepper were used.
Soybean, Wheat, Corn and Mustard:
Leaves that germinated from commercially available seeds of soybean, wheat, corn and mustard were used.
Preparation of Black Pepper Powder Containing Buckwheat Chaff:
0.1 g of ground buckwheat chaff was added to 0.9 g of ground black pepper to obtain black pepper powder containing 10% buckwheat chaff.
(2) Isolation of DNA from Buckwheat, Buckwheat Chaff, Black Pepper, White Pepper and Black Pepper Powder Containing Buckwheat Chaff
DNA isolation was conducted by using the QIAGEN Genomic-tip according to the procedures described in the Genomic DNA Handbook with a few modifications thereto as stated below.
Into a 15 ml-tube was transferred 1 g of a ground sample, added 4 ml of Carlson Lysis Buffer (0.1 M Tris-HCl(pH 9.5), 2% CTAB, 1.4 M Polyethylene Glycol # 6000, 20 mM EDTA), 8 μl of RNase A (100 mg/ml), 10 μl of 2-mercaptoethanol and 80 μl of proteinase K (20 mg/ml) thereto and mixed, and the resulting mixture was incubated for 20 min. at 74° C.
After cooling down to room temperature, to the mixture was added 5 ml of phenol/chloroform/isoamyl alcohol (25/24/1) at room temperature and mixed well by inverting the tube. After centrifuging them, a resulting upper water layer, was collected. The water layer was mixed well with the same volume of chloroform/isoamyl alcohol (24/1) and after centrifuging, a resulting upper water layer was collected. The water layer was mixed well with chloroform/isoamyl alcohol (24/1), and after centrifuging, a resulting water layer was collected again and used in the next step.
Half of the volume of the water layer obtained above was subjected to isopropanol precipitation to collect crude DNA. The collected crude DNA was dissolved in 500 μl of Buffer QBT and the resulting solution was applied to the Genomic-tip 20/G column equilibrated with 1 ml of Buffer QBT to adsorb DNA. Subsequently, the column was washed with 5 ml of Buffer QBT and then with 2 ml of Buffer QC. Finally, DNA was eluted with 1.7 ml of Buffer QF, and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which was then dissolved in 40 μl of sterilized ultrapure water. After the concentration of the resulting DNA preparation was determined, the DNA preparation was used for a PCR template.
(3) Isolation of DNA from Leaves of Wheat, Soybean, Corn and Mustard
DNA isolation was conducted by using the QIAGEN DNeasy Plant Mini Kit according to the procedures described in the DNeasy Plant Mini Kit Handbook mentioned below.
0.5 g of a ground sample was transferred to a 15 ml-tube, added 3 ml of Buffer AP1 and 30 μl of RNase A (100 mg/ml), and mixed well with them. Then the resulting mixture was incubated for 15 min. at 65° C. 975 μl of Buffer AP2 was added to the mixture. The resulting mixture was incubated for 10 min. on ice and then centrifuged to obtain a supernatant. The supernatant was applied to a QIAshredder Spin Column and a flow-through fraction was obtained by centrifuging the column. To the flow-through fraction was added 0.5 volume of Buffer AP3 and 1 volume of ethanol, and mixed. The resulting mixture was divided into halves to be applied to two separate DNeasy Spin Columns. 650 μl of the mixture was applied to a DNeasy Spin Column and the column was centrifuged for 1 min. at 6,000×g to adsorb DNA. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 500 μl of Buffer AW and was centrifuged for 1 min. at 6,000×g. To the column was added 500 μL of Buffer AW again and was centrifuged for 1 min. at a maximum speed to flush out the remaining Buffer AW. Finally, to the column was added 120 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at 6,000×g to obtain a DNA eluate. After the concentration thereof was determined, the DNA eluate was used for a PCR template.
(4) Preparation of DNA Solutions for Evaluation of Sensitivity (Buckwheat in Black Pepper Powder)
A DNA preparation derived from black pepper powder containing 10% buckwheat chaff was diluted stepwise with a DNA preparation from black pepper to obtain black pepper DNA solutions containing 1%, 0.1%, 100 ppm, 10 ppm, 1 ppm, 100 ppb, and 10 ppb of buckwheat chaff DNA. Both DNA preparations used above were obtained according to the procedures described in (2).
(5) Preparation of DNA Solutions for Evaluation of Sensitivity (Buckwheat in Wheat)
A DNA preparation derived from buckwheat seeds was diluted stepwise with a DNA preparation from wheat leaves to obtain wheat leaf DNA solutions containing 1 ppm, 100 ppb, 10 ppb, and 1 ppb of buckwheat seed DNA. The DNA preparation from buckwheat seeds was obtained according to the procedures described in (2). The DNA preparation from wheat leaves was obtained according to the procedures described in (3).
C. PCR
PCR was conducted using the QIAGEN HotStarTaq Master Mix Kit according to the procedures described in the HotStarTaq PCR Handbook as stated below.
PCR was carried out using final volumes of 25 μl of a solution containing 12.5 μl of 2× HotStarTaq Master Mix (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, 400 μM each dNTP), 0.2 μM of each primer (SEQ NO:11 and SEQ NO:3), the template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a GeneAmp PCR System 9600 (Applied Biosystems) according to the following PCR program: pre-incubation at 95° C. for 15 min.; 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min. The PCR reaction mixture was electrophoresed on a 2% agarose gel containing ethidium bromide. After the electrophoresis, the gel was analyzed using a FluorImager 595 (Amersham Pharmacia Biotech). The results are shown in FIGS. 1-3. The meanings of numerical values, abbreviations and symbols used in FIGS. 1-3 are as follows.

    M: 100 bp DNA Ladder Marker
    (−): Negative Control (no DNA)
    Numerical values above sample names: the amounts of the template DNA
    Arrow: indicates the target amplification product (140 bp)
    
        The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of a plant chloroplast DNA.

D. PCR Results

    
    


PCR described above was conducted using primers designed in the present invention. The results are shown in FIGS. 1-3. As shown in FIG. 1, target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat). On the other hand, no 140 bp amplification product was obtained from wheat, mustard, soybean, corn, and white pepper. However, nonspecific amplification products whose sizes were different from the target product were obtained from soybean (approximately 230 bp) and wheat (approximately 2,300 bp). Because these results almost matched the simulation results shown in Table 1, the simulation results were thought to be reliable. Consequently, it was confirmed that a wide range of plants in the genus Fagopyrum including common buckwheat and Tartarian buckwheat were detectable using the present invention.
As shown in FIG. 2, target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from black pepper powder samples containing 10 to 1 ppm of buckwheat chaff. This result showed that buckwheat chaff present in an amount of more than 1 ppm in black pepper is detectable.
As shown in FIG. 3, the target 140 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from the wheat DNA sample containing 10 to 1 ppm of buckwheat DNA.
The nonspecific amplification product that was obviously different from the target product in size did not interfere in the detection of 1 ppm of buckwheat DNA. This result showed that buckwheat DNA present in an amount of more than 1 ppm in wheat DNA is detectable.
E. Preparation of a Sequencing Sample
(1) Purification of the Amplification Product from Buckwheat Chaff
Purification of the amplification product from buckwheat chaff obtained in section D was conducted by using the QIAGEN QIAquick PCR Purification Kit according to the procedures described in the QIAquick Spin Handbook as stated below.
To 1 volume of PCR reaction mixture was added 5 volumes of Buffer PB and mixed well. After being spun down by centrifugation, to a QIAquick Spin Column was applied the mixture and centrifuged for 1 min. at 10,000×g to adsorb DNA. Then, to the column was added 750 μl of buffer PE to wash and centrifuged for 1 min. at 10,000×g. In addition, the column was centrifuged for 1 min. at 10,000×g to remove Buffer PE completely. Finally, to the column was added 50 μl of Buffer EB, let stand for 1 min., and then centrifuged for 1 min. at 10,000×g. The resulting eluted DNA was used for a sequencing sample.
(2) Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides
PCR for sequencing of the purified amplification product from buckwheat chaff obtained in (1) was conducted by using the Applied Biosystems BigDye Terminator Cycle Sequencing FS Ready Reaction Kit according to the procedures described in the manufacturer's manual as stated below.
The PCR for sequencing was carried out using final volumes of 20 μl of a solution containing 8 μl of BigDye Terminator RR Mix, 3.2 μmol of primer (SEQ NO:3), 2 ng template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a GeneAmp PCR System 9600 (Applied Biosystems) according to the following PCR program: pre-incubation at 96° C. for 1 min.; 25 cycles consisting of denaturation at 96° C. for 10 secs. and annealing and extension at 60° C. for 1 min.
Subsequently, removal of the excess dye-labeled dideoxynucleotides from the sequencing reaction mixture was conducted by using the Amersham Pharmacia Biotech AutoSeq G-50 according to the procedures described in the manufacturer's manual as stated below.
The AutoSeq G-50 column was uncapped and 100 μl of 10 mM EDTA was added to the resin in the column. The column was then capped and the resin inside was suspended thoroughly by vortexing. The cap was then loosened and the bottom closure of the column was snapped off. The column was then uncapped and placed in a 2-ml microcentrifuge tube for support and centrifuged for 1 min. at 2,000×g. The column was then placed in a new 2-ml microcentrifuge tube and the sample was applied to the resin in the column. After being capped, the column was centrifuged for 1 min. at 2,000×g, and the flow-through fraction obtained was used in the next step.
F. Sequence Analysis
The sample for sequencing from buckwheat chaff obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of common buckwheat, Fagopyrum esculentum (AB000330), registered in GenBank. The result is shown in FIG. 4. The meanings of number symbols above a nucleotide sequence, lines underneath this sequence and other symbols used in FIG. 4 are described below.

    Number Symbols Nucleotide numbers of amplification products
    Asterisks (*): Identical nucleotides between two nucleotide sequences
    Dash (-): Unidentifiable nucleotide
    S: Mixed nucleotide with C and G
    Single underline: The sense primer region
    Double underline: The antisense primer region

G. The result of Sequence Analysis



As shown in FIG. 4, the amplification product from buckwheat chaff obtained by PCR using the primers designed in the present invention almost matched the sequence of common buckwheat, Fagopyrum esculentum (AB000330). 87 out of 89 bases between two primer regions were determined in a sequence of the product and 86 of these bases matched the sequence of common buckwheat If the base 44, determined as a mixture of two bases, is considered to match the sequence of common buckwheat, all 87 of these bases matched to the sequence of common buckwheat. This result indicates the target ITS-1 ̃5.8S rRNA gene sequence of plants in the genus Fagopyrum can be detected without fail. Furthermore, the source of the buckwheat chaff, used in the aforementioned experiments, was traced back to common buckwheat (Fagopyrum esculentum).
Example 2
A. Design of Oligonucleotide Primers for Detection of DNA from Buckwheat
(1) DNA Sequences of the Genus Fagopyrum, Other Common Allergenic Plants and Plants Widely Used for a Food Ingredient
The DNA sequences described in Example 1 (1) “DNA Sequences of the Genus Fagopyrum”, (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers.
(2) DNA Sequence of Related Species of the Genus Fagopyrum 
As representatives of the DNA sequences of related species of the genus Fagopyrum, 5.8S rRNA gene, 1TS-1 and ITS-2 sequences in the following 27 DNA sequences registered in GenBank were selected. In this connection, the 27 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Fagopyrum, each of which had the highest score in the corresponding genus other than genus Fagopyrum and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-1 sequence of buckwheat (Fagopyrum esculentum AB000330).

    1: Aconogonum sp. Won 152 (AF189731)
    2: Fallopia scandens (AF040069)
    3: Polygonum virginianum (U51274)
    4: Rumex acetosella (AF189730)
    5: Talinum paraguayense (L78056)
    6: Bruinsmia styracoides (AF396438)
    7: Talinella pachypoda (L78054)
    8: Rehderodendron kwangtungense (AF396448)
    9: Pterostyrax corymbosus (AF396445)
    10: Anredera cordifolia (L78086)
    11: Cistanthe quadripetala (L78062)
    12: Xenia vulcanensis (L78060)
    13: Talinopsis frutescens (L78058)
    14: Talinaria palmeri (L78052)
    15: Portulaca sp. (L78049)
    16: Phemeranthus confertiflorus (L78039)
    17: Montiopsis umbellata (L78033)
    18: Grahamia bracteata (L78028)
    19: Herniaria glabra (AJ310965)
    20: Alluaudia duwosa (L78011)
    21: Sinojackia xylocarpa (AF396451)
    22: Halesia macgregori (AF396442)
    23: Changiostyrax dolichocarpa (AF396439)
    24: Alectryon subdentatus (AF314765)
    25: Anacampseros recurvata (L78014)
    26: Weinmannia racemosa (AF485597)
    27: Bursera tecomaca (AF080029)

(3) Oligonucleotide Primer Synthesis and Evaluation



Among ITS-1 sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum, there was determined a nucleotide sequence which would specifically hybridize to all of the 21 DNA sequences of the genus Fagopyrum and would not induce nonspecific amplification products from soybean through the study of the ITS-1 sequences. The thus determined nucleotide sequence is indicated as SEQ NO:14. Subsequently, the oligonucleotide primer with SEQ NO:14 was synthesized.









Sense primer




5′-CGC CAA GGA CCA CGA ACA GAA G-3′
(SEQ NO:14)






The oligonucleotide primer with SEQ NO:3 was also used as an antisense primer, the same as in Example 1.
Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1. As a result, it was predicted that target 146 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. In contrast, obtaining of any 146 bp amplification product was not predicted to be obtained from the aforementioned 8 DNA sequences of other common allergenic plants (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown Lice, pepper and mustard) and the 2 DNA sequences among related species of the genus Fagopyrum belonging to Polygonaceae and the 23 DNA sequences of related species of the genus Fagopyrum not belonging to Polygonaceae. In this connection, the results of simulation indicated some possibility that an amplification product, whose size almost matched the target product size of 146 bp, would be obtained from the sequences of Aconogonum sp. Won 152 and Fallopia scandens in the related species of the genus Fagopyrum belonging to Polygonaceae. However, by sequence analysis of the amplification products, it is possible to identify either the genus Fagopyrum or the related species thereof. The simulation results are shown in Tables 2A to 2C. The meanings of symbols and numerical values in Tables 2A to 2C are explained below.

    ★: An obtained amplification product whose size almost matched the target product size 146 bp (±10 bp).
    W 2-6: Probability of obtaining amplification products
    
        High Probability—W6>W5>W4>W3>W—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Fagopyrum: 
    
        Sequences similar to the ITS-1 sequence of Fagopyrum esculentum (AB000330) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Table 2C as the representative of the DNA sequences of related species of the genus Fagopyrum.
    
    









TABLE 2A







Buckwheat, SEQ NO: 14 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Fagopyrum
★Fagopyrum urophyllum
AB000342
146 bp
—
439 bp
—
—



★Fagopyrum urophyllum
AB000341
146 bp
—
—
—
—



★Fagopyrum tataricum
AB000340
146 bp
—
—
—
—



(Tartarian buckwheat)



★Fagopyrum tataricum
AB000339
146 bp
—
—
—
—



(Tartarian buckwheat)



★Fagopyrum statice
AB000338
146 bp
—
—
—
—



★Fagopyrum statice
AB000337
146 bp
—
—
—
—



★Fagopyrum pleioramosum
AB000336
146 bp
—
—
—
—



★Fagopyrum lineare
AB000335
—
146 bp
—
—
—



★Fagopyrum leptopodum
AB000334
146 bp
—
—
—
—



★Fagopyrum homotropicum
AB000333
146 bp
—
—
—
—



★Fagopyrum gracilipes
AB000332
146 bp
—
—
—
—



★Fagopyrum esculentum
AB000331
146 bp
—
—
—
—



(Common buckwheat)



★Fagopyrum esculentum
AB000330
146 bp
—
—
—
—



(Common buckwheat)



★Fagopyrum cymosum
AB000329
146 bp
—
—
—
—



★Fagopyrum cymosum
AB000328
146 bp
—
—
—
—



★Fagopyrum cymosum
AB000327
146 bp
—
—
—
—



★Fagopyrum cymosum
AB000326
146 bp
—
—
—
—



★Fagopyrum cymosum
AB000325
146 bp
—
—
—
—



★Fagopyrum cymosum
AB000324
146 bp
—
—
—
—



★Fagopyrum capillatum
AB000323
146 bp
—
—
—
—



★Fagopyrum callianthum
AB000322
146 bp
—
439 bp
—
—
















TABLE 2B







Buckwheat, SEQ NO: 14 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Other Common Allergenic
Arachis hypogaea (Peanut)
AF156675
—
—
—
—
—


Plants
Triticum aestivum (Wheat)
AJ301799
—
—
—
—
—



Glycine max (Soybean)
U60551
—
—
—
—
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
—



Malus x domestica (Apple)
AF186484
—
—
—
—
—



Citrus sp. (Valencia orange)
E08821
—
—
—
—
—


Plants Widely Used for a Food
Zea mays (Corn)
U46648
—
—
—
—
—


Ingredient
Oryza sativa (Brown rice)
AF169230
—
—
—
—
—



Piper nigrum (Pepper)
AF275197
—
—
—
—
—



Sinapis alba (Mustard)
X15915
—
—
—
—
—
















TABLE 2C







Buckwheat, SEQ NO: 14 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus
★Aconogonum sp. Won 152
AF189731
—
149 bp
—
—
—


Fagopyrum Belonging to
★Fallopia scandens
AF040069
—
145 bp
—
—
—


Plygonaceae
Polygonum virginianum
U51274
—
—
—
—
—



Rumex acetosella
AF189730
—
—
—
—
—


Related Species of the Genus Fagopyrum
Talinum paraguayense
L78056
—
—
—
—
—


Not Belonging to Polygonaceae
Bruinsmia styracoides
AF396438
—
—
—
—
—



Talinella pachypoda
L78054
—
—
—
—
—



Rehderodendron kwangtungense
AF396448
—
—
—
—
—



Pterostyrax corymbosus
AF396445
—
—
—
—
—



Anredera cordifolia
L78086
—
—
—
—
—



Cistanthe quadripetala
L78062
—
—
—
—
—



Xenia vulcanensis
L78060
—
—
—
—
—



Talinopsis frutescens
L78058
—
—
—
—
—



Talinaria palmeri
L78052
—
—
—
—
—



Portulaca sp.
L78049
—
—
—
—
—



Phemeranthus confertiflorus
L78039
—
—
—
—
—



Montiopsis umbellata
L78033
—
—
—
—
—



Grahamia bracteata
L78028
—
—
—
—
—



Herniaria glabra
AJ310965
—
—
—
—
—



Alluaudia dumosa
L78011
—
—
—
—
—



Sinojackia xylocarpa
AF396451
—
—
—
—
—



Halesia macgregori
AF396442
—
—
—
—
—



Changiostyrax dolichocarpa
AF396439
—
—
—
—
—



Alectryon subdentatus
AF314765
—
—
—
—
—



Anacampseros recurvata
L78014
—
—
—
—
—



Weinmannia racemosa
AF485597
—
—
—
—
—



Bursera tecomaca
AF080029
—
—
—
—
—









B. Preparation of Template DNA for PCR

The DNA samples isolated from buckwheat, pepper, wheat, soybean, corn and mustard in Examples 1B (2) and (3) and the DNA solutions for evaluation of sensitivity prepared in Example 1B (5) were used.
C. PCR
PCR was conducted in the same way as in Example 1C, except for the use of the following primer and PCR program.
Primer:
Each primer of SEQ NO:14 and SEQ NO:3 was used at 0.2 μM of final concentration.
PCR Program:
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 mm.
The results are shown in FIGS. 5 and 6. The meanings of numerical values, abbreviations and symbols used in FIGS. 5 and 6 are as follows.

    M: 100 bp DNA Ladder Marker
    (−): Negative control (no DNA)
    Numerical values above sample names: the amounts of the template DNA
    Arrow: indicating the target amplification product (146 bp)
    
        The quality of each of the template DNAs used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA.

D. PCR Results

    
    


PCR described above was conducted using primers designed in the present invention. The results are shown in FIGS. 5 and 6. As shown in FIG. 5, target 146 bp amplification products, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, were obtained from Shirahana soba (common buckwheat) and Dattan soba (Tartarian buckwheat). On the other hand, no 146 bp amplification product was obtained from wheat, mustard, soybean, corn, and white pepper (nonspecific amplification obtained from soybean in Example 1 was not obtained either). Likewise, no 146 bp amplification product was obtained from brown rice (not shown in FIG. 5). However, nonspecific amplification products whose sizes were different from that of the target product were obtained from wheat (approximately 2,300 bp). Because these results almost matched the simulation results shown in Table 2, the simulation results were thought to be reliable. Consequently, it was confirmed that a wide range of plants in the genus Fagopyrum including shirahana soba (common buckwheat) and Tartarian buckwheat were detectable using the present invention.
As shown in FIG. 6, the target 140 bp amplification product, predicted from the simulation results of the 21 DNA sequences of the genus Fagopyrum, was obtained from the wheat DNA sample containing 10 to 1 ppm of buckwheat DNA.
The nonspecific amplification product that was obviously different from the target in size did not interfere in the detection of 1 ppm of buckwheat DNA. This result showed that buckwheat DNA present in an amount of more than 1 ppm in wheat DNA is detectable.
E. Preparation of a Sequencing Sample
(1) Purification of the Amplification Product from Shirahana Soba (Common Buckwheat)
Purification of the amplification product from Shirahana soba (common buckwheat) obtained in D was conducted in the same way as in Example 1E

    (1) “Purification of the Amplification Product from Buckwheat Chaff”.
    (2) Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides


PCR for sequencing of the purified amplification product from Shirahana soba (common buckwheat) obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for the use of primers of SEQ NO:14 and SEQ NO:3).
F. Sequence Analysis
The sample for sequencing from Shirahana soba (common buckwheat) obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of common buckwheat, Fagopyrum esculentum (AB000331) and F. homotropicum (AB000340) in GenBank. The result is shown in FIG. 7. The meanings of number symbols above a nucleotide sequence, lines underneath this sequence and other symbols used in FIG. 7 are described below.

    Number Symbols: Nucleotide numbers of amplification products
    Asterisks (*): Nucleotides of Shirahana soba (common buckwheat), which are identical with those of both nucleotide sequences of F. esculentum (AB000331) and F. homotropicum (AB000340)
    *: Nucleotides of Shirahana soba (common buckwheat), which are identical with those of only one of the nucleotide sequences of F. esculentum (AB000331) and F. homotropicum (AB000340)
    Single underline The sense primer region
    Double underline The antisense primer region

G. The result of Sequence Analysis



As shown in FIG. 7, all 89 bases between two primer regions in the amplification product derived from Shirahana soba (common buckwheat) by PCR using the primers designed in the present invention are completely identical with one of sequences of common buckwheat, F. esculentum (AB000331) or F. homotropicum (AB000340). This result indicates the target ITS-1 ̃5.8S rRNA gene sequence of plants in the genus Fagopyrum can be detected without fail. Furthermore, the source of the Shirahana soba (common buckwheat), used in the aforementioned experiments, was traced back to Fagopyrum esculentum or F. homotropicum. 
Example 3
A. Design of Oligonucleotide Primers for Detection of DNA from Peanut
(1) DNA Sequences of the Genus Arachis 
Regarding the genus Arachis, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 11 DNA sequences registered in GenBank were examined to select suitable regions for the primers.

    1: Arachis batizocoi (AF203553)
    2: Arachis correntina (AF203554)
    3: Arachis hermannii (AF203556)
    4: Arachis hoehnei (AJ320395)
    5: Arachis hypogaea (AF156675)
    6: Arachis magna (AF203555)
    7: Arachis major (AF203552)
    8: Arachis palustris (AF203557)
    9: Arachis pintoi (AF203551)
    10: Arachis triseminata (AF204233)
    11: Arachis villosa (AF203558)

(2) DNA Sequences of Other Common Allergenic Plants



The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequence registered in GenBank were also selected.

    1: buckwheat: Fagopyrum esculentum (AB000330)

(3) DNA Sequences of Plants Widely Used for a Food Ingredient



The DNA sequences described in Example 1A (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected.
(4) DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient
Regarding French bean, lima bean, lentil, chickpea, mung bean and adzuki bean, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected. In the case of adzuki bean, only the ITS-1 sequence of Vigna angularis var. nipponensis (AB059747) was selected because the 5.8S rRNA gene sequence was not registered in GenBank.

    1: French bean: Phaseolus vulgaris (AF115169)
    2: lima bean: Phaseolus lunatus (AF115175)
    3: lentil: Lens culinaris subsp. culinaris (AF228066)
    4: chickpea: Cicer arietinum (AJ237698)
    5: mung bean: Vigna radiata (X14337)
    6: adzuki bean: Vigna angularis var. nipponensis (AB059747)

(5) DNA Sequences of Plants in Related Species of the Genus Arachis



As representatives of the DNA sequences of related species of the genus Arachis, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 69 DNA sequences registered in GenBank were selected. In this connection, the 69 DNA sequences were selected as representatives of the DNA sequence of related species of the genus Arachis, each of which had the highest score in the corresponding genus other than genus Arachis and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from ITS-1 sequence of buckwheat through BLAST homology search. (Arachis hypogaea AF 156675)

    1: Stylosanthes acuminata (AJ320282)
    2: Stylosanthes angustifolia (AJ320284)
    3: Stylosanthes aurea (AJ320285)
    4: Stylosanthes biflora (AJ320289)
    5: Stylosanthes bracteata (AJ320346)
    6: Stylosanthes calcicola (AJ320348)
    7: Stylosanthes campestris (AJ320291)
    8: Stylosanthes capitata (AJ320350)
    9: Stylosanthes cayennensis (AJ320292)
    10: Stylosanthes erects (AJ320352)
    11: Stylosanthes fruticosa (AJ320356)
    12: Stylosanthes gracilis (AJ320296)
    13: Stylosanthes grandifolia (AJ320299)
    14: Stylosanthes guianensis subsp. dissitiflora (AJ320301)
    15: Stylosanthes hamata (AJ320365)
    16: Stylosanthes hippocampoides (AJ320317)
    17: Stylosanthes hispida (AJ320328)
    18: Stylosanthes humilis (AJ320323)
    19: Stylosanthes ingrata (AJ320329)
    20: Stylosanthes leiocarpa (AJ320332)
    21: Stylosanthes linearifolia (AJ320367)
    22: Stylosanthes macrocarpa (AJ320369)
    23: Stylosanthes macrocephala (AJ320371)
    24: Stylosanthes macrosoma (AJ320333)
    25: Stylosanthes mexicana (AJ320374)
    26: Stylosanthes montevidensis (AJ320336)
    27: Stylosanthes pilosa (AJ320377)
    28: Stylosanthes scabra (AJ320382)
    29: Stylosanthes seabrana (AJ320384)
    30: Stylosanthes sericeiceps (AJ320386)
    31: Stylosanthes subsericea (AJ320387)
    32: Stylosanthes sundaica (AJ320389)
    33: Stylosanthes sympodialis (AJ320391)
    34: Stylosanthes tomentosa (AJ320337)
    35: Stylosanthes tuberculata (AJ320392)
    36: Stylosanthes viscosa (AJ320340)
    37: Ormocarpum bernierianum (AF189036)
    38: Ormocarpum coeruleum (AF189037)
    39: Ormocarpum drakei (AF189039)
    40: Ormocarpum flavum (AF189041)
    41: Ormocarpum keniense (AF068155)
    42: Ormocarpum kirkii (AF068152)
    43: Ormocarpum klainei (AF189044)
    44: Ormocarpum megalophyllum (AF068154)
    45: Ormocarpum muricatum (AF068156)
    46: Ormocarpum orientale (AF068159)
    47: Ormocarpum pubescens (AF189045)
    48: Ormocarpum rectangulare (AF189046)
    49: Ormocarpum schliebenii (AfF189047)
    50: Ormocarpum sennoides (AF068153)
    51: Ormocarpum somalense (AF 189048)
    52: Ormocarpum trachycarpum (AF189049)
    53: Ormocarpum trichocarpum (AF068158)
    54: Ormocarpum verrucosum (AF189050)
    55: Chapmannia floridana (AF203543)
    56: Chapmannia prismatica (AJ320400)
    57: Chapmannia somalensis (AF203544)
    58: Ormocarpopsis aspera (AF068148)
    59: Ormocarpopsis calcicola (AF068145)
    60: Ormocarpopsis itremoensis (AF068149)
    61: Ormocarpopsis mandrarensis (AF068147)
    62: Ormocarpopsis parvifolia (AF068144)
    63: Ormocarpopsis tulearensis (AF068146)
    64: Diphysa humilis (AF068162)
    65: Diphysa macrophylla (AF189029)
    66: Diphysa suberosa (AF189034)
    67: Spigelia coelostylioides (AF177992)
    68: Spigelia hedyotidea (AF 178005)
    69: Spigelia marilandica (AF177991)

(6) Oligonucleotide Primer Synthesis and Evaluation



Among ITS-1 sequences of the aforementioned 11 DNA sequences of the genus Arachis, there was determined three nucleotide sequences which would specifically hybridize to all of the 11 DNA sequences of the genus Arachis through the study of the ITS-1 sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:18, 19 and 20. Subsequently, the oligonucleotide primers with SEQ NOs:18, 19 and 20 were synthesized.











Sense primers





5′-GCG GAA AGC GCC AAG GAA GC-3′
(SEQ NO:18)



 



5′-CGG CTT CCG GAG ACG GCA-3′
(SEQ NO:19)



 



5′-CGG CTC CGG AGA CGG CA-3′
(SEQ NO:20)






As an antisense primer, the oligonucleotide primer with SEQ NO:3 was also used, the same as in Example 1.
Regarding the sense and antisense primer pairs, a simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), in the same manner as in Example 1. As a result, it was predicted that 156 to 157 bp (a combination of the primers with SEQ NOs:18 and 3), 114 to 116 bp (a combination of the primers with SEQ NOs:19 and 3) and 113 to 115 bp (a combination of the primers with SEQ NOs:20 and 3) of target amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis. 
Furthermore, regarding the sense and antisense primer pairs, it was predicted whether amplification products would be obtained from the aforementioned 8 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, rise, pepper and mustard) and the 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean).
Regarding the combination of the primers with SEQ NOs:18 and 3, the result of a simulation indicated that desired amplification products having almost 156 bp would not be obtained from 7 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown rice, pepper and mustard) and the 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean). In this connection, the results of the simulation indicated some possibility that amplification products having almost 156 bp would be obtained from apple from among the other common allergenic plants. However, by a sequence analysis of the amplification products, it is possible to identify either peanut or apple. The simulation results are shown in Tables 3A and 3B. The meanings of symbols and numerical values in Tables 3A and 3B are explained below.

    ★: An obtained amplification product whose size almost matched the target product size 156 bp (±10 bp).
    W 2-6: Probability of obtaining amplification products
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    (−*): No annealing site of the primer (SEQ NO: 18) was predicted within the ITS-1 sequence of Vigna angularis var. nipponensis (adzuki bean).
    
        In the case of adzuki bean, only the ITS-1 sequence was selected because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.
    
    









TABLE 3A







Peanut, SEQ NO: 18 & SEQ NO: 3 primer: Amplification products













Scientific Name
GenBank







(Common Name)
Accession No.
W6
W5
W4
W3
W2

















Genus Arachis
★Arachis batizocoi
AF203553
156 bp
—
—
—
—



★Arachis correntina
AF203554
156 bp
—
—
—
—



★Arachis hermannii
AF203556
156 bp
—
—
—
—



★Arachis hoehnei
AJ320395
156 bp
—
—
—
—



★Arachis hypogaea
AF156675
156 bp
—
—
—
—



(Peanut)



★Arachis magna
AF203555
156 bp
—
—
—
—



★Arachis major
AF203552
156 bp
—
—
—
—



★Arachis palustris
AF203557
156 bp
—
—
—
—



★Arachis pintoi
AF203551
157 bp
—
—
—
—



★Arachis triseminata
AF204233
156 bp
—
—
—
—



★Arachis villosa
AF203558
156 bp
—
—
—
—


Other Common Allergenic Plants
Fagopyrum esculentum
AB000330
—
—
—
—
—



(Common buckwheat)



Triticum aestivum
AJ301799
—
—
—
—
—



(Wheat)



Glycine max
U60551
—
—
—
—
—



(Soybean)



Juglans regia
AF303809
—
—
—
—
—



(Walnut)



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica
AF185621
—
—
—
—
—



(Peach)



★Malus x domestica
AF186484
—
155 bp
—
424 bp
—



(Apple)




467 bp



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)
















TABLE 3B







Peanut, SEQ No: 18 & SEQ No: 3 primer: Amplification products













Scientific Name
GenBank







(Common Name)
Accession No.
W6
W5
W4
W3
W2

















Plants Widely Used
Zea mays
U46648
—
—
—
—
—


for a Food Ingredient
(Corn)



Oryza sativa
AF169230
—
—
—
—
—



(Brown rice)



Piper nigrum
AF275197
—
—
—
—
—



(Pepper)



Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Leguminous Plants Widely Used for a
Phaseolus vulgaris
AF115169
—
—
—
—
—


Food Ingredient
(French bean)



Phaseolus lunatus
AF115175
—
—
—
—
—



(Lima bean)



Lens culinaris subsp.
AF228066
—
—
—
—
—



culinaris



(Lentil)



Cicer arietinum
AJ237698
—
—
—
—
—



(Chickpea)



Vigna radiata
X14337
—
—
—
—
—



(Mung bean)











Vigna angularis var.
AB059747
—*



nipponensis



(Adzuki bean)*










Regarding the combination of the primers with SEQ NOs:20 and 3, no amplification product having almost 114 bp was predicted from 6 DNA 5 sequences of common allergenic plants other than peanut (wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used as a food ingredient (corn, brown lice, pepper and mustard) and the 5 out of 6 DNA sequences of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea and mung bean). In this connection, the results of the simulation indicated some possibility that nonspecific amplification products having almost 114 bp would be obtained from buckwheat and from soybean from among the other common allergenic plants and from adzuki bean in the leguminous plants widely used for a food ingredient in even weak amplified signals. Here, regarding the DNA sequence of adzuki bean (Vigna angulans var. nipponensis AB059747), as the ITS-1 sequence was registered in GenBank, but the 5.8S rRNA gene sequence was not registered therein, the amplification product having almost 100 bp was estimated based on the predicted annealing site of SEQ NO: 20 to the ITS-1 sequence and the assumption that Vigna angulans var. nipponensis (adzuki bean) had the same 5.8S rRNA gene sequence as Arachis hypogaea (peanut) and the primer with SEQ NO: 3 had an annealing site within the 5.8S rRNA gene sequence. However, despite the prediction of amplification, the probability of obtaining these amplification products compared with obtaining the target amplification product of the genus Arachis were lower than the probability of obtaining amplification for peanut, and by a sequence analysis of the amplification products, it is also possible to identify whether they are peanut or not.
In addition, regarding the combination of the primers with SEQ NOs:20 and 3, no amplification products having almost 100 bp were predicted from the 69 DNA sequences of related species of both the genus Arachis belonging to leguminous plants and those not belonging to leguminous plants. The simulation results are shown in Tables 4A to 4E. The meanings of symbols and numerical values in Tables 4A to 4E are explained below.
As to the simulation in which SEQ NO: 19 and SEQ NO: 3 were used, the result is not shown here because it was later found that this primer pair was not suitable for PCR analysis to detect the genus Arachis. 

    ★: An obtained amplification product whose size almost matched the target 114 bp (±10 bp).
    W 2-6: Probability of Obtaining Amplification Products
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Arachis: 
    
        Sequences similar to the ITS-1 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected among them. Each sequence having highest score in each genus and having a score of 60 bits or more is shown in the following Tables 4D-4E as the representatives of the DNA sequences of related species of the genus Arachis. 
    
    
    (+*): An annealing site of the primer (SEQ NO: 20) was predicted within the ITS-1 of Vigna angularis var. nipponensis (adzuki bean).
    
        In the case of adzuki bean, only the ITS-1 sequence registered in GenBank was used because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank. Furthermore, the size of the amplification product (approximately 100 bp) was estimated based on the predicted annealing site of the SEQ NO: 20 within the ITS-1 sequence and the assumption described below. It was assumed that Vigna angularis var. nipponensis (adzuki bean) had the same 5.8S rRNA gene sequence as Arachis hypogaea (peanut) and the primer with the SEQ NO: 3 had an annealing site within the 5.8S rRNA gene sequence.
    
    









TABLE 4A







Peanut, SEQ NO: 20 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Arachis
★Arachis batizocoi
AF203553
114 bp
—
281 bp
—
52 bp



★Arachis correntina
AF203554
114 bp
—
282 bp
—
—



★Arachis hermannii
AF203556
114 bp
—
281 bp
—
—



★Arachis hoehnei
AJ320395
114 bp
—
284 bp
—
—



★Arachis hypogaea
AF156675
—
113 bp
286 bp
—
—



(Peanut)



★Arachis magna
AF203555
114 bp
—
282 bp
—
—



★Arachis major
AF203552
114 bp
—
281 bp
—
—



★Arachis palustris
AF203557
114 bp
—
282 bp
—
—



★Arachis pintoi
AF203551
115 bp
—
283 bp
—
—



★Arachis triseminata
AF204233
114 bp
—
281 bp
—
—



★Arachis villosa
AF203558
114 bp
—
283 bp
—
—


Other Common Allergenic Plants
★Fagopyrum esculentum
AB000330
—
—
109 bp
—
—



(Common buckwheat)



Triticum aestivum
AJ301799
—
—
247 bp
—
—



(Wheat)



154 bp



Glycine max
U60551
—
—
 98 bp
—
—



(Soybean)



Juglans regia
AF303809
—
—
—
—
—



(Walnut)



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica
AF185621
—
—
—
—
—



(Peach)



Malus x domestica
AF186484
—
—
—
—
—



(Apple)



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)
















TABLE 4B







Peanut, SEQ NO: 20 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Plants Widely Used
Zea mays
U46648
—
—
—
—
—


for a Food Ingredient
(Corn)



Oryza sativa
AF169230
—
—
—
—
—



(Brown rice)



Piper nigrum
AF275197
—
—
—
—
80 bp



(Pepper)



Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Leguminous Plants Widely Used
Phaseolus vulgaris
AF115169
—
—
—
—
—


for a Food Ingredient
(French bean)



Phaseolus lunatus
AF115175
—
—
—
—
—



(Lima bean)



Lens culinaris subsp.
AF228066
—
—
—
—
—



Culinaris



(Lentil)



Cicer arietinum
AJ237698
—
—
—
—
—



(Chickpea)



Vigna radiata
X14337
—
—
—
—
—



(Mung bean)











★Vigna angularis var.
AB059747
+* (approximately 100 bp)



nipponensis



(Adzuki bean)*

















TABLE 4C







Peanut, SEQ NO: 20 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Stylosanthes acuminata
AJ320282
—
—
—
—
—


Belonging to Leguminous Plants
Stylosanthes angustifolia
AJ320284
—
—
—
—
—



Stylosanthes aurea
AJ320285
—
—
—
—
—



Stylosanthes biflora
AJ320289
—
—
—
—
—



Stylosanthes bracteata
AJ320346
—
—
—
—
—



Stylosanthes calcicola
AJ320348
—
—
—
—
—



Stylosanthes campestris
AJ320291
—
—
—
—
—



Stylosanthes capitata
AJ320350
—
—
217 bp
351 bp
—








384 bp



Stylosanthes cayennensis
AJ320292
—
—
—
—
—



Stylosanthes erecta
AJ320352
—
—
—
—
—



Stylosanthes fruticosa
AJ320356
—
—
—
—
—



Stylosanthes gracilis
AJ320296
—
—
—
—
—



Stylosanthes grandifolia
AJ320299
—
—
—
—
—



Stylosanthes guianensis subsp.
AJ320301
—
—
—
—
—



dissitiflora



Stylosanthes hamata
AJ320365
—
—
—
—
—



Stylosanthes hippocampoides
AJ320317
—
—
—
—
—



Stylosanthes hispida
AJ320328
—
—
—
—
—



Stylosanthes humilis
AJ320323
—
—
—
—
—



Stylosanthes ingrata
AJ320329
—
—
—
—
—



Stylosanthes leiocarpa
AJ320332
—
—
—
—
—
















TABLE 4D







Peanut, SEQ NO: 20 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Stylosanthes linearifolia
AJ320367
—
—
—
—
—


Belonging to Leguminous Plants
Stylosanthes macrocarpa
AJ320369
—
—
—
—
—



Stylosanthes macrocephala
AJ320371
—
—
217 bp
384 bp
—



Stylosanthes macrosoma
AJ320333
—
—
—
—
—



Stylosanthes mexicana
AJ320374
—
—
—
—
—



Stylosanthes montevidensis
AJ320336
—
—
—
—
—



Stylosanthes pilosa
AJ320377
—
—
—
—
—



Stylosanthes scabra
AJ320382
—
—
—
—
—



Stylosanthes seabrana
AJ320384
—
—
—
—
—



Stylosanthes sericeiceps
AJ320386
—
—
—
—
—



Stylosanthes subsericea
AJ320387
—
—
—
—
—



Stylosanthes sundaica
AJ320389
—
—
—
—
—



Stylosanthes sympodialis
AJ320391
—
—
—
—
—



Stylosanthes tomentosa
AJ320337
—
—
—
—
—



Stylosanthes tuberculata
AJ320392
—
—
—
—
—



Stylosanthes viscosa
AJ320340
—
—
—
—
—



Ormocarpum bernierianum
AF189036
—
—
—
—
—



Ormocarpum coeruleum
AF189037
—
—
—
—
—



Ormocarpum drakei
AF189039
—
—
—
—
—



Ormocarpum flavum
AF189041
—
—
—
—
—



Ormocarpum keniense
AF068155
—
—
213 bp
—
—



Ormocarpum kirkii
AF068152
—
—
—
—
—



Ormocarpum klainei
AF189044
—
—
—
—
—
















TABLE 4E







Peanut, SEQ NO: 18 & SEQ NO: 3 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Ormocarpum megalophyllum
AF068154
—
—
—
—
—


Belonging to Leguminous Plants
Ormocarpum muricatum
AF068156
—
—
260 bp
—
—



Ormocarpum orientale
AF068159
—
—
—
—
—



Ormocarpum pubescens
AF189045
—
—
215 bp
—
—



Ormocarpum rectangulare
AF189046
—
—
—
—
—



Ormocarpum schliebenii
AF189047
—
—
—
—
—



Ormocarpum sennoides
AF068153
—
—
—
—
—



Ormocarpum somalense
AF189048
—
—
—
—
—



Ormocarpum trachycarpum
AF189049
—
—
214 bp
—
—



Ormocarpum trichocarpum
AF068158
—
—
—
—
—



Ormocarpum verrucosum
AF189050
—
—
—
—
—



Chapmannia floridana
AF203543
—
—
—
—
—



Chapmannia prismatica
AJ320400
—
—
—
—
—



Chapmannia somalensis
AF203544
—
—
—
—
—



Ormocarpopsis aspera
AF068148
—
—
—
—
—



Ormocarpopsis calcicola
AF068145
—
—
—
—
—



Ormocarpopsis itremoensis
AF068149
—
—
—
—
—



Ormocarpopsis mandrarensis
AF068147
—
—
—
—
—



Ormocarpopsis parvifolia
AF068144
—
—
—
—
—



Ormocarpopsis tulearensis
AF068146
—
—
—
—
—



Diphysa humilis
AF068162
—
—
—
—
—



Diphysa macrophylla
AF189029
—
—
—
—
—



Diphysa suberosa
AF189034
—
—
—
—
—


Related Species of the Genus
Spigelia coelostylioides
AF177992
—
—
—
—
—


Arachis Not Belonging to
Spigelia hedyotidea
AF178005
—
—
—
—
—


Leguminous Plants
Spigelia marilandica
AF177991
—
—
—
—
—









B. Preparation of Template DNA for PCR

(1) Samples Used for DNA Extraction

Peanut:

6 commercially available peanuts were used.
Buckwheat, Wheat, Soybean, Adzuki Bean and Corn:
Leaves that germinated from commercially available seeds of Shirahana soba (common buckwheat), wheat, 2 soybeans, 2 adzuki beans and corn were used.
(2) Isolation of DNA from Peanut
DNA isolation was conducted by using the QIAGEN Genomic-tip and the resulting isolate was purified by MACHEREY-NAGEL NucleoSpin as stated below.
Into a 15 ml-tube 1 g of a ground sample was transferred, added 10 ml of Buffer G2, 100 μl of Proteinase K (20 mg/ml) and 10 μl of RNase A (100 mg/ml), and they were mixed. The resulting mixture was incubated for 1 hour at 50° C. Then the resulting mixture was centrifuged for 10 min. at 3,000×g to obtain a supernatant. The resulting supernatant was applied to the Genomic-tip 20/G column equilibrated with 1 ml of Buffer QBT to adsorb DNA to the column. Subsequently, the column was washed with 4 ml of Buffer QC and DNA was eluted with 1 ml of preheated (50° C.) Buffer QF. To the eluate was added 4 volume of Buffer NT2, mixed with it, and then the resulting mixture was divided into two halves to be applied to two separate NucleoSpin Extract Columns. 650 μl of the mixture was applied to a NucleoSpin Extract Column and then the column was centrifuged for 1 min. at 6,000×g to adsorb DNA to the column. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 600 μl of Buffer NT3 and was centrifuged for 1 min. at 6,000×g. To the column was added 600 μl of Buffer NT3 again and was centrifuged for 1 min. at a maximum speed to flush out the Buffer NT3 remaining in the column. Finally, to the column was added 100 μl of Buffer NE and was centrifuged for 1 min. at a maximum speed to obtain a DNA eluate from the column and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which were then dissolved in 40 μl of sterilized ultrapure water. After the concentration of the resulting DNA preparation was determined, the DNA preparation was used for a PCR template.
(3) Isolation of DNA from Leaves of Shirahana Soba (Common Buckwheat), Wheat, Soybean, Adzuki Bean and Corn
DNA isolation was conducted by using the QIAGEN DNeasy Plant Mini Kit according to the procedures described in the DNeasy Plant Mini Kit Handbook as shown below.
Into a 1.5 ml-tube 50 mg of a ground sample was transferred, added 600 μl of Buffer AP1 and 6 μl of RNase A (100 mg/ml), and they were mixed well. Then the resulting mixture was incubated for 1 hour at 65° C. 2001cl of Buffer AP2 was then added to the mixture. The resulting mixture was incubated for 10 min. on ice and then centrifuged to obtain a supernatant. The resulting supernatant was applied to a QIAshredder Spin Column and a flow-through fraction was obtained by centrifuging the column To the flow-through fraction was added 0.5 volume of Buffer AP3 and 1 volume of ethanol, and mixed with them. The resulting mixture was divided into two halves to be applied to two DNeasy Spin Columns. 650 μl of the mixture was applied to a DNeasy Spin Column and the column was centrifuged for 1 min. at 6,000×g to adsorb DNA to the column. This step was repeated with the remaining mixture. In order to wash the column, to the column was added 500 μl of Buffer AW and was centrifuged for 1 min. at 6,000×g. To the column was added 500 μl of Buffer AW again and was centrifuged for 1 min. at a maximum speed to flush out the Buffer AW remaining in the column. Finally, to the column was added 100 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at a maximum speed, and added another 100 μl of preheated (65° C.) Buffer AE and was centrifuged for 1 min. at a maximum speed to obtain a DNA eluate from the column, and the resulting eluate was subjected to isopropanol precipitation to collect DNA, which were then dissolved in 50 μl of sterilized ultrapure water. After the concentration of the resulting DNA preparation was determined, the DNA eluate was used for a PCR template.
(4) Preparation of DNA Solutions for Evaluation of Sensitivity (Peanut in Wheat)
A DNA preparation derived from peanut seeds was diluted stepwise with a DNA preparation from wheat leaves to obtain wheat leaf DNA solutions containing 10 ppm and 1 ppm of peanut seed DNA. The DNA preparation from peanut seeds was obtained according to the procedures described in (2). The DNA preparation from wheat leaves was obtained according to the procedures described in (3).
C. PCR (Part 1: a combination of primers with SEQ NOs:18 and 3)
PCR was conducted using the Applied Biosystems AmpliTaq Gold(R) & 10×PCR Buffer II & MgCl2 Solution with dNTP as stated below.
PCR was carried out using final volumes of 25 μl of a solution containing 2.5 μl of 10×PCR Buffer II, 0.125 μl of AmpliTaq Gold (5 U/μl), 2.5 μl of dNTPs Mix (2 mM each), 1.5 μl of MgCl2 Solution (25 mM), 0.5 μM of each primer (SEQ NO:18 and SEQ NO:3), the template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a GeneAmp PCR System 2400 (Applied Biosystems) according to the following PCR program: pre-incubation at 95° C. for 15 min.; 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min. The PCR reaction mixture was electrophoresed on a 2% agarose gel containing ethidium bromide. After the electrophoresis, the gel was analyzed using a FluorImager 595 (Amersham Pharmacia Biotech). The results are shown in FIG. 8. The meanings of numerical values, abbreviations and symbols used in FIG. 8 are as follows.

    M: 100 bp DNA Ladder Marker
    (−): Negative control (no DNA)
    Numerical values above sample names: the amounts of the template DNA
    Arrow: indicates the target amplification product (156 bp)

D. PCR Results (Part 1: a combination of primers with SEQ NOs:18 and 3)



PCR described above was conducted using primers designed in the present invention. The results are shown in FIG. 8. As shown in FIG. 8, target 156 bp amplification products, predicted from the simulation results of ITS-1 ̃5.8S rRNA gene sequences of the genus Arachis, were obtained from 6 commercially available peanuts. Because this result almost matched the simulation results shown in Table 3, the simulation results were thought to be reliable.
E. Preparation of a Sequencing Sample (Part 1: a combination of primers with SEQ NOs:18 and 3)
(1) Purification of the Amplification Product from Peanut
A purification of the amplification product from peanut obtained in D was conducted in the same way as in Example 1E (1) “Purification of the Amplification Product from Buckwheat Chaff”.
(2) Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides
PCR for sequencing of the purified amplification product from peanut obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for use of primers with SEQ NOs: 18 and 3.
F. Sequence Analysis (Part 1: a combination of primers with SEQ NOs:18 and 3)
The sample for sequencing from peanut obtained in E (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of the genus Arachis, Arachis hypogaea (AF156675), A. correntina (AF203554) and A. villosa (AF203558) in GenBank. The result is shown in FIG. 9. The meanings of number symbols above a nucleotide sequence, lines underneath a sequence and other symbols used in FIG. 9 are described below.

    Number Symbols: Nucleotide numbers of amplification products
    Asterisks (*): Nucleotides of the peanuts, which are identical with those of all of the nucleotide sequences of Arachis hypogaea (AF156675), A. correntina (AF203554) and A. villosa (AF203558) in GenBank
    Single underline: The sense primer region
    Double underline: The antisense primer region

G. The result of Sequence Analysis (Part 1: a combination of primers with SEQ NOs:18 and 3)



As shown in FIG. 9, the amplification products derived from all of the 6 commercially available peanuts obtained by PCR using the primers designed in the present invention completely (100%) matched from one another, and both the sequence of A. correntina (AF203554) and that of A. villosa (AF156675) completely matched 114 bases between two primer regions. This result indicates the target ITS-1 ̃5.8S rRNA gene sequence of the genus Arachis can be detected without fail. In this connection, the target amplification product differed from A. hypogaea (AF156675) at 5 positions, particularly the 48th base “T” thereof was not present in the genus Arachis except for A. hypogaea (AF156675) in GenBank and it is not present in the commercially available peanuts experimented on here. It was found that, in order to design widely detectable primers to the genus Arachis, which includes commercially available peanuts, a DNA sequence of commercially available peanuts, A. correntina (AF203554) or A. villosa (AF203558), was preferably used in place of a DNA sequence of A. hypogaea (AF156675) in GenBank, and therefore, that the sense primer with SEQ NO:20 designed from the sequence of commercially available peanuts, A. correntina (AF203554) or A. villosa (AF203558) was used rather than the sense primer with SEQ NO:19 designed from the sequence of A. hypogaea (AF156675). H. PCR (Part 2: a combination of primers with SEQ NOs:20 and 3)
PCR was conducted using the QIAGEN HotStarTaq Master Mix Kit according to the procedures described in the HotStarTaq PCR Handbook as stated below.
PCR was carried out using final volumes of 25 μl of a solution containing 12.5 μl of 2× HotStarTaq Master Mix (HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, 400 μM each dNTP), 0.5 μM of each primer (SEQ NO:20 and SEQ NO:3), the template DNA and sterilized ultrapure water in 0.2-ml microcentrifuge tubes. Amplification was performed using a Sequence Detection System ABI PRISM 7700 (Applied Biosystems) according to the following PCR program: pre-incubation at 95° C. for 15 min.; 25 cycles consisting of denaturation at 95° C. for 30 secs., annealing and extension at 75° C. for 30 secs. respectively and 30 cycles consisting of denaturation at 95° C. for 30 secs., annealing and extension at 72° C. for 30 secs. respectively; followed by a final extension at 72° C. for 5 min. The resulting PCR reaction mixture was electrophoresed on a 2% agarose gel containing ethidium bromide. After the electrophoresis, the gel was analyzed using a FluorImager 595 (Amersham Pharmacia Biotech). The results are shown in FIGS. 10 and 11. The meanings of numerical values, abbreviations and symbols used in FIGS. 10 and 11 are as follows.

    M: 100 bp DNA Ladder Marker
    (−): Negative control (no DNA)
    Numerical values above sample names: the amounts of the template DNA
    Arrow: indicates the target amplification product (114 bp)


The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA.
I. PCR Results (Part 2: a combination of primers with SEQ NOs:20 and 3)
PCR described above was conducted using primers designed in the present invention. The results are shown in FIGS. 10 and 11. As shown in FIG. 10, target 114 bp amplification products, predicted from the simulation results of the ITS-1 ̃5.8S rRNA gene sequence of the genus Arachis, were obtained from peanuts. On the other hand, no 114 bp amplification product was obtained from buckwheat, wheat, soybean, adzuki bean and corn. However, nonspecific amplification products, which were different from the target product in size, were obtained from wheat (approximately 250 bp). Because these results almost matched the simulation results shown in Table 4, the simulation results were thought to be reliable. Consequently, it was confirmed that a wide range of plants in the genus Arachis including commercially available peanuts were detectable using the present invention.
As shown in FIG. 11, the target 114 bp amplification product, predicted from the simulation results of the ITS-1 ̃5.8S rRNA gene sequence of the genus Arachis, was obtained from the wheat DNA sample containing 1 ppm of peanut DNA. This result showed that even where 10 to 1 ppm of peanut DNA is present in wheat DNA, the peanut can be detectable.
Furthermore, the nonspecific amplification product from wheat that was obviously different from the target product in size did not interfere in the detection of 1 ppm of peanut DNA.
In this connection, as shown in Examples 1, 2 and 3, where a W value, which shows a possibility of obtaining amplification products in a simulation by Amplify, is not more than W4, it has been found that the amplification products, which differ from the target one, are obtained in some cases and not obtained in other cases in actual PCR. Please note that Tables 1 to 4 show the data of the simulation results of Amplify which are of W2 value or higher but do not show those of a lower W value such as W1 and W0 wherein the possibility of obtaining the amplification products is considered to be low.
J. Preparation of a Sequencing Sample (Part 2: a Combination of Primers with SEQ NOs:20 and 3)
(1) Purification of the Amplification Product from Peanut
The purification of the amplification product from peanut obtained in I was conducted in the same way as in Example 1E (1) “Purification of the Amplification Product from Buckwheat Chaff”.
(2) Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides
PCR for sequencing of the purified amplification product from peanut obtained in (1) was conducted in the same way as in Example 1E (2) “Sequencing PCR Reaction and Removal of Excess Dye-Labeled Dideoxynucleotides”, except for use of primers with SEQ NOs:20 and 3.
K. Sequence Analysis (Part 2: A Combination of Primers with SEQ NOs:20 and 3)
The sample for sequencing from peanut obtained in J (2) was analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence of the amplification product was compared with the sequence of Arachis hypogaea (AF156675), A. correntina (AF203554), A. villosa (AF203558), A. major (AF203552) and A. hermannii (AF203556) in the genus Arachis and the nucleotide sequence of the commercially available peanut obtained in G. The result is shown in FIG. 12. The meanings of number symbols above a nucleotide sequence, lines underneath this sequence and other symbols used in FIG. 12 are described below.

    Number Symbols: Nucleotide numbers of amplification products
    Commercially available peanuts: A part of the nucleotide sequence of the commercially available peanuts obtained in G
    Asterisks (*): Nucleotides of the peanuts, which are identical with those of all of the sequences of Arachis hypogaea (AF156675), A. correntina (AF203554), A. villosa (AF203558), A. major (AF203552) and A. hermannii (AF203556) in the genus Arachis and the sequence of the commercially available peanut obtained in G.
    Single underline: The sense primer region
    Double underline: The antisense primer region

L. The Result of Sequence Analysis (Part 2: a Combination of Primers with SEQ NOs:20 and 3)



As shown in FIG. 12, a part of the nucleotide sequence of an amplification product derived from commercially available peanuts obtained by PCR using the primers designed in the present invention completely (100%) matched 75 bases between two primer regions of respective sequences of Arachis correntina (AF203554), A. villosa (AF203558Y, A. Major (AF203552) and A. hermannii (AF203556). In this connection, the target amplification product differed from Arachis hypogaea (AF156675) at 4 positions, but completely (100%) matched the nucleotide sequence of the commercially available peanuts obtained in G. This result indicates the target ITS-1 ̃5.8S rRNA gene sequence of the genus Arachis can be detected without fail.
Example 4
A. Design of Oligonucleotide Primers for Detection of DNA from Buckwheat
(1) DNA Sequences of the Genus Fagopyrum, Other Common Allergenic Plants and Plants Widely Used for a Food Ingredient
The DNA sequences described in Example 1A (1) “DNA sequences of the Genus Fagopyrum”, (2) “DNA Sequences of other common allergenic plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers.
(2) DNA Sequence of Plants in Related Species of the Genus Fagopyrum 
The DNA sequences described in Example 2A (2) “DNA Sequence of Plants in Related Species of the Genus Fagopyrum” were selected.
(3) Oligonucleotide Primer Synthesis
Among ITS-1 sequences of the aforementioned 21 DNA sequences of the genus Fagopyrum, there was determined a nucleotide sequence which would specifically hybridize to all of the 21 DNA sequences of the genus Fagopyrum through the study of the ITS-1 sequences. The thus determined nucleotide sequence is indicated as SEQ NO:15. Subsequently, the oligonucleotide primer with SEQ NO:15 was synthesized.









Sense primer




5′-CGT TGC CGA GAG TCG TTC TGT TT-3′
(SEQ NO:15)






The oligonucleotide primer with SEQ NO:14 was also used as an antisense primer, the same as in Example 2.
B. PCR Simulation
Regarding the sense and antisense primer pair, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1, to examine whether a target size of amplification products would be obtained from the 21 DNA sequences of the genus Fagopyrum, the 8 DNA sequences of common allergenic plants other than buckwheat (peanut, wheat, soybean, walnut, matsutake mushroom, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard) and the 27 sequences of related species of the genus Fagopyrum. 
The simulation results are shown in Tables 5A and 5C. The meanings of symbols and numerical values in Tables 5A and 5C are explained below.

    ★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
    W 2-6: Probability of Obtaining Amplification Products
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Fagopyrum: 


Sequences similar to the ITS-1 sequence of Fagopyrum esculentum (AB000330) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 5B-5C as the representative of the DNA sequences of related species of the genus Fagopyrum. 
C. Preparation of Template DNA for PCR
The DNA samples isolated from Shirahana soba (common buckwheat) and Dattan soba (Tartalian buckwheat) in Example 1B (2) were diluted stepwise with sterilized ultrapure water to use them. The DNA samples isolated from white pepper in Example 1B (2), mustard in Example 1B (3), peanut in Example 3B (2) and wheat, soybean, and corn in Example 3B (3) were also used. In addition, the DNA samples isolated from brown lice seeds and Fallopia convolvulus in the same way as in Example 1B (3) were used. The DNA samples from Fallopia convolvulus were diluted stepwise with sterilized ultrapure water to use.
D. PCR
PCR was conducted in essentially the same way as in Example 1C, except for use of the following primers and PCR program.
Primers:
Each primer of SEQ NO:14 and SEQ NO:15 was used at 0.5 μM of a final concentration.
PCR Program:
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 2 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
E. Results
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to the ITS-1 ̃5.8S rRNA gene sequence of each plant. As a result, as shown in Tables 5A to 5C, it was predicted that target 101 bp amplification products would be obtained from the aforementioned 21 DNA sequences of the genus Fagopyrum. On the other hand, it was predicted that no target 101 bp amplification product would be obtained from the 8 DNA sequences of other common allergenic plants (peanut, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard) and the 27 DNA sequences of related species of the genus Fagopyrum both belonging to Polygonaceae and not belonging to Polygonaceae. Also, no nonspecific amplification product could be predicted. As a result, it was confirmed that a wide range of plants in the genus Fagopyrum would be specifically detectable using the present invention.







TABLE 5A







Buckwheat, SEQ No: 14 & SEQ No: 15 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Fagopyrum
★Fagopyrum urophyllum
AB000342
101 bp
—
439 bp
—
—



★Fagopyrum urophyllum
AB000341
101 bp
—
—
—
—



★Fagopyrum tataricum
AB000340
101 bp
—
—
—
—



(Tartarian buckwheat)



★Fagopyrum tataricum
AB000339
101 bp
—
—
—
—



(Tartarian buckwheat)



★Fagopyrum statice
AB000338
101 bp
—
—
—
—



★Fagopyrum statice
AB000337
101 bp
—
—
—
—



★Fagopyrum pleioramosum
AB000336
101 bp
—
—
—
—



Fagopyrum lineare
AB000335
101 bp
—
—
—
—



★Fagopyrum leptopodum
AB000334
101 bp
—
—
—
—



★Fagopyrum homotropicum
AB000333
101 bp
—
—
—
—



Fagopyrum gracilipes
AB000332
101 bp
—
—
—
—



★Fagopyrum esculentum
AB000331
101 bp
—
—
—
—



(Common buckwheat)



★Fagopyrum esculentum
AB000330
101 bp
—
—
—
—



(Common buckwheat)



★Fagopyrum cymosum
AB000329
101 bp
—
—
—
—



★Fagopyrum cymosum
AB000328
101 bp
—
—
—
—



★Fagopyrum cymosum
AB000327
101 bp
—
—
—
—



★Fagopyrum cymosum
AB000326
101 bp
—
—
—
—



★Fagopyrum cymosum
AB000325
101 bp
—
—
—
—



★Fagopyrum cymosum
AB000324
101 bp
—
—
—
—



★Fagopyrum capillatum
AB000323
101 bp
—
—
—
—



★Fagopyrum callianthum
AB000322
101 bp
—
440 bp
—
—
















TABLE 5B







Buckwheat, SEQ No: 14 & SEQ No: 15 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Other Common Allergenic Plants
Arachis hypogaea
AF156675
—
—
—
—
—



(Peanut)



Triticum aestivum
AJ301799
—
—
—
—
—



(Wheat)



Glycine max
U60551
—
—
—
—
—



(Soybean)



Juglans regia
AF303809
—
—
—
—
—



(Walnut)



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica
AF185621
—
—
—
—
—



(Peach)



Malus x domestica
AF186484
—
—
—
—
—



(Apple)



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)


Plants Widely Used
Zea mays
U46648
—
—
—
—
—


for a Food Ingredient
(Corn)



Oryza sativa
AF169230
—
—
—
—
—



(Brown rice)



Piper nigrum
AF275197
—
—
—
—
—



(Pepper)



Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Related Species of the Genus
Aconogonum sp. Won 152
AF189731
—
—
—
—
—


Fagopyrum Belonging to
Fallopia scandens
AF040069
—
—
—
—
—


Polygonaceae
Polygonum virginianum
U51274
—
—
—
—
—



Rumex acetosella
AF189730
—
—
—
—
—
















TABLE 5C







Buckwheat, SEQ No: 14 & SEQ No: 15 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Fagopyrum
Talinum paraguayense
L78056
—
—
—
—
—


Not Belonging to Polygonaceae
Bruinsmia styracoides
AF396438
—
—
—
—
—



Talinella pachypoda
L78054
—
—
—
—
—



Rehderodendron
AF396448
—
—
—
—
—



kwangtungense



Pterostyrax corymbosus
AF396445
—
—
—
—
—



Anredera cordifolia
L78086
—
—
—
—
—



Cistanthe quadripetala
L78062
—
—
—
—
—



Xenia vulcanensis
L78060
—
—
—
—
—



Talinopsis frutescens
L78058
—
—
—
—
—



Talinaria palmeri
L78052
—
—
—
—
—



Portulaca sp.
L78049
—
—
—
—
—



Phemeranthus
L78039
—
—
—
—
—



confertiflorus



Montiopsis umbellata
L78033
—
—
—
—
—



Grahamia bracteata
L78028
—
—
—
—
—



Herniaria glabra
AJ310965
—
—
—
—
—



Alluaudia dumosa
L78011
—
—
—
—
—



Sinojackia xylocarpa
AF396451
—
—
—
—
—



Halesia macgregori
AF396442
—
—
—
—
—



Changiostyrax dolichocarpa
AF396439
—
—
—
—
—



Alectryon subdentatus
AF314765
—
—
—
—
—



Anacampseros recurvata
L78014
—
—
—
—
—



Weinmannia racemosa
AF485597
—
—
—
—
—



Bursera tecomaca
AF080029
—
—
—
—
—









PCR described above was conducted using primers designed in the present invention. In this case, target 101 bp amplification products, predicted from the simulation results of the ITS-1 ̃5.8S rRNA gene sequences of the genus Fagopyrum, were obtained from 500 to 50 fg of Shirahana soba (common buckwheat) DNA and Dattan soba (Tartarian buckwheat) DNA. As a result, it was found that even where 500 to 50 fg of buckwheat DNA is present, the buckwheat can be detected. In this connection, such sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of buckwheat DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples. On the other hand, no amplification product having 101 bp or nonspecific amplification products was obtained from wheat, peanut, soybean, corn, mustard, pepper and brown rice. Furthermore, regarding Fallopia convolvulus, when an amount of template DNA is 50 to 5 ng, a target size of an amplification product was obtained with a very weak signal, but when an amount of template DNA is 500 pg or less, no amplification product in a target size or nonspecific amplification product was obtained at all. In this connection, regarding Fallopia convolvulus, when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples, even if 1% of Fallopia convolvulus was present in sample DNA, the level of Fallopia convolvulus DNA corresponds to a non-detected level as false positive. By modification of PCR program, there is a possibility that amplification products in a target size will not be obtained from 50 to 5 ng of DNA from Fallopia convolvulus. 
Consequently, in conjunction with the results of specificity studied by PCR simulation and of sensitivity and specificity studied by PCR, it was confirmed that a wide range of the genus Fagopyrum including common buckwheat and Tartarian buckwheat were detectable using the present invention.
Example 5
A. Design of Oligonucleotide Primers for Detection of DNA from Peanut
(1) DNA Sequences of the Genus Arachis, Other Common Allergenic Plants, Plants Widely Used for a Food Ingredient and Leguminous Plants Widely Used for a Food Ingredient
The DNA sequences described in Example 3A (1) “DNA Sequences of the Genus Arachis”, Example 3A (2) “DNA Sequences of Other Common Allergenic Plants”, Example 3A (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” and Example 3A (4) “DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient” were examined to select suitable regions for the primers. In addition, as a DNA sequence of adzuki bean, 5.8S rRNA gene, ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: adzuki bean: Vigna angularis vars nipponensis (AB060088)

(2) DNA Sequences of Plants in Related Species of the Genus Arachis 



As representatives of the DNA sequences of related species of the genus Arachis, respective 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 45 DNA sequences registered in GenBank were examined to select suitable regions for the primers. In this connection, the 45 DNA sequences were selected as representations, each of which was of highest Score in the species which were other than peanut (Arachis hypogaea AF156675) and were of Score of 60 bits or more among sequences of said species hit to ITS-2 sequence of peanut through BLAST homology search.

    1: Chapmannia floridana (AF203543)
    2: Chapmannia gracilis (AF203546)
    3: Chapmannia prismatica (AJ320400)
    4: Chapmannia reghidensis (AF204232)
    5: Chapmannia sericea (AF203548)
    6: Chapmannia somalensis (AF203544)
    7: Chapmannia tinireana (AF203547)
    8: hebrigiella gracilis (AF203561)
    9: Fissicalyx fendleri (AF189061)
    10: Stylosanthes acuminata (AJ320282)
    11: Stylosanthes angustifolia (AJ320284)
    12: Stylosanthes aurea (AJ320285)
    13: Stylosanthes biflora (AJ320289)
    14: Stylosanthes bracteata (AJ320346)
    15: Stylosanthes calcicola (AJ320348)
    16: Stylosanthes campestris (AJ320291)
    17: Stylosanthes capitata (AJ320350)
    18: Stylosanthes cayennensis (AJ320292)
    19: Stylosanthes erecta (AJ320352)
    20: Stylosanthes fruticosa (AJ320356)
    21: Stylosanthes gracilis (AJ320296)
    22: Stylosanthes grandifolia (AJ320299)
    23: Stylosanthes guianensis subsp. dissitiflora (AJ320301)
    24: Stylosanthes hamata (AJ320365)
    25: Stylosanthes hippocampoides (AJ320316)
    26: Stylosanthes hispida (AJ320328)
    27: Stylosanthes humilis (AJ320327)
    28: Stylosanthes ingrata (AJ320329)
    29: Stylosanthes leiocarpa (AJ320332)
    30: Stylosanthes linearifola (AJ320367)
    31: Stylosanthes macrocarpa (AJ320369)
    32: Stylosanthes macrocephala (AJ320371)
    33: Stylosanthes macrosoma (AJ320333)
    34: Stylosanthes mexicana (AJ320373)
    35: Stylosanthes montevidensis (AJ320336)
    36: Stylosanthes pilosa (AJ320377)
    37: Stylosanthes scabra (AJ320382)
    38: Stylosanthes seabrana (AJ320384)
    39: Stylosanthes sericeiceps (AJ320386)
    40: Stylosanthes subsericea (AJ320387)
    41: Stylosanthes sundaica (AJ320389)
    42: Stylosanthes sympodialis (AJ320391)
    43: Stylosanthes tomentosa (AJ320337)
    44: Stylosanthes tuberculata (AJ320392)
    45: Stylosanthes viscosa (AJ320340)


In addition, if PCR simulation is conducted by selecting a primer hybridized to ITS-1 sequence, the DNA sequences described in Example 3A (5) “DNA Sequences of Plants in Related Species to the Genus Arachis” were also selected.
(3) Oligonucleotide Primer Synthesis
(a) Among 5.8S rRNA gene sequences of the aforementioned 11 DNA sequences of the genus Arachis and the aforementioned 8 DNA sequences of other common allergenic plants, there was determined nucleotide sequences which would hybridize to all DNA sequences of these plants through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:7. Subsequently, the oligonucleotide primer with SEQ NO:7 was synthesized.










Sense primer









(SEQ NO:7)











5′-GAT GAA GAA CGT AGC GAA ATG CGA TAC T-3′







Among ITS-2 sequences of the aforementioned 11 DNA sequences of the genus Arachis, there was determined nucleotide sequences which would specifically hybridize to all of the 11 DNA sequences of the genus Arachis through the study of the sequences. The thus determined nucleotide sequence is indicated as SEQ NO:24. Subsequently, the oligonucleotide primer with SEQ NO:24 was synthesized.











Antisense primer





5′-CCA TCT GCC GCG GTG CC-3′
(SEQ NO:24)






(b) Furthermore, in another combination of primers, the primer of SEQ NO:18 described in Example 3 was selected as sense primer on the ITS-1 sequence and the primer of SEQ NO:24 was selected as antisense primer on the ITS-2 sequence.

B. PCR Simulation

Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 11 DNA sequences of the genus Arachis, the 8 DNA sequences of common allergenic plants other than peanut (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the DNA sequences of plants in related species of the genus Arachis. 
(a) The simulation results using a combination of primers of SEQ NOs:7 and 24 are shown in Tables 6A to 6D. The meanings of symbols and numerical values in Tables 6A to 6D are explained below.


    ★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
    W 2-6: Probability to obtain amplification products 
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Arachis: 
    
        Sequences similar to the ITS-2 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or one were selected among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables GB-6D as the representative of the DNA sequences of related species of the genus Arachis. 
    
    
    (−*): No annealing site of the primer (SEQ NO: 24) was predicted within the ITS-2 sequence of Vigna angularis var. nipponensis (adzuki bean).
    
        In the case of adzuki bean, only the ITS-2 sequence was selected because the 5.8S rRNA gene sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.

(b) The simulation results using a combination of primers of SEQ NOs:18 and 24 are shown in Tables 7A to 7E. The meanings of symbols and numerical values in Tables 7A to 7E are explained below.

    
    
    ★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
    W 2-6: Probability to obtain amplification products 
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Arachis: 
    
        Sequences Similar to the ITS-1 or ITS-2 sequence of Arachis hypogaea (AF156675) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 7B-7E as the representative of the DNA sequences of related species of the genus Arachis. 
    
    
    (−*): No annealing site of the primer (SEQ NO:18) was predicted within the ITS-1 sequence of Vigna angularis var. nipponensis (adzuki bean) and no annealing site of the primer (SEQ NO:24) was predicted within the ITS-2 sequence of Vigna angularis var. nipponensis (adzuki bean).


In the case of adzuki bean, only either the ITS-1 or ITS-2 sequence was selected respectively because full length of the ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequence of Vigna angularis var. nipponensis (AB059747) was not registered in GenBank.
C. Preparation of Template DNA for PCR
A DNA preparation from peanut isolated in Example 3B (2) was diluted stepwise with sterilized ultrapure water to use.
D. PCR
PCR was conducted in substantially the same way as in Example 1C, except for use of following primers and PCR program.
Primer:


    (a) Each primer of SEQ NO:7 and SEQ NO:24 was used at 0.5 μM of final concentration.
    (b) Each primer of SEQ NO:18 and SEQ NO:24 was used at 0.5, M of final concentration.

PCR Program:



PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA.
E. Results
(a) Results of the Combination of the Primers of SEQ NOs:7 and 24
Regarding the primers of the present invention (the combination of the primers of SEQ NOs:7 and 24), the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to 5.8S rRNA gene ̃ITS-2 sequence of each plants. As a result, as shown in Tables GA to GD, it was predicted that target 253 to 259 bp amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis. On the other hand, it was predicted that no target 253 to 259 bp amplification products would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 41 DNA sequences of plants in related species of the genus Arachis belonging to leguminous plants. In this connection, among the plants in related species of the genus Arachis belonging to leguminous plants, it was predicted by simulation that amplification products having almost 253 to 259 bp would be obtained from the DNA sequences of Stylosanthes cayennensis, Stylosanthes hispida, Stylosanthes viscosa and Fissicalyx fendleri, but these amplification products can be identified by sequence analysis. Optionally, whether these amplification products are peanut may be also identified by PCR showing in Example 3 and the like. As a result, it was confirmed that wide ranges of plants in the genus Arachis would be specifically detectable using the present invention.







TABLE 6A







Peanut, SEQ NO: 7 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Arachis
★Arachis batizocoi
AF203553
—
253 bp
318 bp
214 bp
—







149 bp



★Arachis correntina
AF203554
—
254 bp
323 bp
219 bp
—







150 bp



★Arachis hermannii
AF203556
—
253 bp
320 bp
216 bp
—







149 bp



★Arachis hoehnei
AJ320395
—
256 bp
152 bp
—
—



★Arachis hypogaea
AF156675
—
259 bp
968 bp
222 bp
69 bp



(Peanut)



941 bp







328 bp







175 bp







153 bp



★Arachis magna
AF203555
—
254 bp
322 bp
218 bp
—







150 bp



★Arachis major
AF203552
—
253 bp
320 bp
216 bp
—







149 bp



★Arachis palustris
AF203557
—
254 bp
323 bp
219 bp
—







150 bp



★Arachis pintoi
AF203551
—
254 bp
323 bp
219 bp
—







150 bp



★Arachis triseminata
AF204233
—
253 bp
149 bp
—
—



★Arachis villosa
AF203558
—
255 bp
324 bp
220 bp
—







151 bp


Other Common Allergenic Plants
Fagopyrum esculentum
AB000330
—
—
—
160 bp
—



(Common buckwheat)



Triticum aestivum (Wheat)
AJ301799
—
—
—
—
—



Glycine max (Soybean)
U60551
—
—
—
—
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
—



Malus x domestica
AF186484
—
—
—
—
—



(Apple)



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)
















TABLE 6B







Peanut, SEQ NO: 7 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Plants Widely Used
Zea mays
U46648
—
—
—
—
 82 bp


for a Food
(Corn)


Ingredient
Oryza sativa
AF169230
—
—
—
—
 99 bp



(Brown rice)





 92 bp



Piper nigrum
AF275197
—
—
—
—
—



(Pepper)



Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Leguminous Plants Widely Used
Phaseolus vulgaris
AF115169
—
—
—
—
—


for a Food Ingredient
(French bean)



Phaseolus lunatus
AF115175
—
—
—
—
—



(Lima bean)



Lens culinaris subsp.
AF228066
—
—
—
—
 98 bp



culinaris (Lentil)



Cicer arietinum
AJ237698
—
—
—
—
—



(Chickpea)



Vigna radiata
X14337
—
—
—
—
—



(Mung bean)











Vigna angularis var.
AB060088
—*



nipponensis



(Adzuki bean)*














Related Species of the Genus
Chapmannia floridana
AF203543
—
—
330 bp
35 bp
226 bp


Arachis Belonging to leguminous
Chapmannia gracilis
AF203546
—
—
—
—
—


Plants
Chapmannia prismatica
AJ320400
—
—
—
—
—



Chapmannia reghidensis
AF204232
—
—
—
—
—



Chapmannia sericea
AF203548
—
—
—
—
—



Chapmannia somalensis
AF203544
—
—
—
—
—



Chapmannia tinireana
AF203547
—
—
—
—
—



Fiebrigiella gracilis
AF203561
—
—
—
—
—



★Fissicalyx fendleri
AF189061
—
—
252 bp
—
349 bp







170 bp

340 bp









258 bp









148 bp









 66 bp
















TABLE 6C







Peanut, SEQ NO: 7 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Stylosanthes acuminata
AJ320282
—
—
—
—
—


Belonging to Leguminous Plants
Stylosanthes angustifolia
AJ320284
—
—
—
—
—



Stylosanthes aurea
AJ320285
—
—
—
—
—



Stylosanthes biflora
AJ320289
—
—
—
—
—



Stylosanthes bracteata
AJ320346
—
—
—
—
—



Stylosanthes calcicola
AJ320348
—
—
—
—
—



Stylosanthes campestris
AJ320291
—
—
—
—
—



Stylosanthes capitata
AJ320350
—
—
—
—
—



★Stylosanthes cayennensis
AJ320292
—
253 bp
455 bp
—
372 bp







170 bp



Stylosanthes erecta
AJ320352
—
—
—
—
—



Stylosanthes fruticosa
AJ320356
—
—
—
—
—



Stylosanthes gracilis
AJ320296
—
—
—
—
—



Stylosanthes grandifolia
AJ320299
—
—
—
—
—



Stylosanthes guianensis
AJ320301
—
—
—
—
—



subsp. dissitiflora



Stylosanthes hamata
AJ320365
—
—
—
—
—



Stylosanthes
AJ320316
—
—
—
—
—



hippocampoides



★Stylosanthes hispida
AJ320328
—
253 bp
455 bp
—
372 bp







170 bp



Stylosanthes humilis
AJ320327
—
—
170 bp
—
 66 bp
















TABLE 6D







Peanut, SEQ NO: 7 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Stylosanthes ingrata
AJ320329
—
—
—
—
—


Belonging to Leguminous Plants
Stylosanthes leiocarpa
AJ320332
—
—
175 bp
—
71 bp



Stylosanthes linearifolia
AJ320367
—
—
—
—
—



Stylosanthes macrocarpa
AJ320369
—
—
—
—
—



Stylosanthes macrocephala
AJ320371
—
—
—
—
—



Stylosanthes macrosoma
AJ320333
—
—
—
—
—



Stylosanthes mexicana
AJ320373
—
—
—
—
—



Stylosanthes
AJ320336
—
—
—
—
—



montevidensis



Stylosanthes pilosa
AJ320377
—
—
—
—
—



Stylosanthes scabra
AJ320382
—
—
—
—
—



Stylosanthes seabrana
AJ320384
—
—
—
—
—



Stylosanthes sericeiceps
AJ320386
—
—
—
—
—



Stylosanthes subsericea
AJ320387
—
—
—
—
—



Stylosanthes sundaica
AJ320389
—
—
—
—
—



Stylosanthes sympodialis
AJ320391
—
—
—
—
—



Stylosanthes tomentosa
AJ320337
—
—
—
—
—



Stylosanthes tuberculata
AJ320392
—
—
—
—
—



★Stylosanthes viscosa
AJ320340
—
436 bp
149 bp
—
—






253 bp









PCR described above was conducted using primers designed in the present invention. In this case, target 253 to 259 bp amplification products, expected from the simulation results of the 5.8S rRNA gene ̃ITS-2 sequences of the genus Arachis, were obtained from 500 to 50 fg of peanut DNA. It is found from the results that even where 500 to 50 fg of peanut DNA is present, the peanut can be detected. In this connection, this sensitivity correspond to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and of sensitivity and specificity studied by PCR, it was confirmed that a wide range of the genus Arachis including peanut were detectable using the present invention.
(b) Results of the Combination of the Primers of SEQ NOs:18 and 24
Regarding the primers of the present invention (the combination of the primers of SEQ NOs:18 and 24), the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequence of each plant. As a result, as shown in Tables 7A to 7E, it was predicted that target 384 to 390 bp amplification products would be obtained from the aforementioned 11 DNA sequences of the genus Arachis. On the other hand, it was predicted that no target 384 to 390 bp amplification products would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, wheat, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown lice, pepper and mustard), the 7 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean), the 71 DNA sequences of plants in related species of the genus Arachis belonging to leguminous plants and the 3 DNA sequences of plants in related species of the genus Arachis not belonging to leguminous plants. Among the plants in related species of the genus Arachis belonging to leguminous plants, it was predicted by simulation that amplification products, which were almost matched 384 to 390 bp of the target one in size, would be obtained from the DNA sequences of Stylosanthes cayennensis, Stylosanthes hispida, Stylosanthes viscosa and Fissicalyx fendleri, but these amplification products can be identified by sequence analysis. Optionally, whether these amplification products are peanut may be also identified by PCR showing in Example 3 and the like. As a result, it was confirmed that wide ranges of plants in the genus Arachis would be specifically detectable using the present invention.







TABLE 7A







Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Arachis
★Arachis batizocoi
AF203553
—
384 bp
149 bp
214 bp
—






449 bp
 72 bp



★Arachis correntina
AF203554
—
385 bp
150 bp
219 bp
—






454 bp



★Arachis hermannii
AF203556
—
384 bp
149 bp
216 bp
—






451 bp



★Arachis hoehnei
AJ320395
—
387 bp
152 bp
—
—







 60 bp



★Arachis hypogaea
AF156675
—
390 bp
306 bp
1099 bp 
 69 bp



(Peanut)


459 bp
153 bp
1072 bp 








222 bp



★Arachis magna
AF203555
—
385 bp
150 bp
218 bp
—






453 bp
 72 bp



★Arachis major
AF203552
—
384 bp
149 bp
216 bp
—






451 bp



★Arachis palustris
AF203557
—
385 bp
150 bp
219 bp
—






454 bp
 72 bp



★Arachis pintoi
AF203551
—
386 bp
150 bp
219 bp
—






455 bp



★Arachis triseminata
AF204233
—
384 bp
149 bp
—
—



★Arachis villosa
AF203558
—
386 bp
151 bp
220 bp
—






455 bp


Other Common Allergenic Plants
Fagopyrum esculentum
AB000330
—
—
—
160 bp
—



(Common buckwheat)



Triticum aestivum
AJ301799
—
—
—
—
—



(Wheat)



Glycine max (Soybean)
U60551
—
—
—
—
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
—



★Malus x domestica
AF186484
—
—
—
467 bp
—



(Apple)




424 bp



Citrus sp.
E08821
—
—
—
103 bp
280 bp



(Valencia orange)
















TABLE 7B







Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Plants Widely Used
Zea mays
U46648
—
—
—
—
82 bp


for a Food
(Corn)


Ingredient
Oryza sativa
AF169230
—
—
—
—
99 bp



(Brown rice)





92 bp



Piper nigrum
AF275197
—
—
—
—
—



(Pepper)



Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Leguminous Plants Widely Used
Phaseolus vulgaris
AF115169
—
—
—
—
—


for a Food Ingredient
(French bean)



Phaseolus lunatus
AF115175
—
—
—
—
—



(Lima bean)



Lens culinaris subsp.
AF228066
—
—
—
—
98 bp



culinaris (Lentil)



Cicer arietinum
AJ237698
—
—
—
—
95 bp



(Chickpea)



Vigna radiata (Mung bean)
X14337
—
—
—
—
—











Vigna angularis var.
AB059747
—*



nipponensis



(Adzuki bean)*



Vigna angularis var.
AB060088
—*



nipponensis



(Adzuki bean)*














Related Species of the Genus Arachis
Stylosanthes acuminata
AJ320282
—
—
—
—
—


Belonging to Leguminous
Stylosanthes angustifolia
AJ320284
—
—
—
—
—



Stylosanthes aurea
AJ320285
—
—
—
—
—



Stylosanthes biflora
AJ320289
—
—
—
—
—



Stylosanthes bracteata
AJ320346
—
—
—
—
—



Stylosanthes calcicola
AJ320348
—
—
—
—
—



Stylosanthes campestris
AJ320291
—
—
—
—
—



Stylosanthes capitata
AJ320350
—
—
—
—
—



★Stylosanthes cayennensis
AJ320292
—
384 bp
455 bp
301 bp
372 bp 



Stylosanthes erecta
AJ320352
—
—
—
—
—



Stylosanthes fruticosa
AJ320356
—
—
—
—
—



Stylosanthes gracilis
AJ320296
—
—
—
—
—



Stylosanthes grandifolia
AJ320299
—
—
—
—
—
















TABLE 7C







Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Stylosanthes guianensis
AJ320301
—
—
—
—
—


Belonging to Leguminous
subsp. dissitiflora



Stylosanthes hamata
AJ320365
—
—
—
—
—



Stylosanthes
AJ320317
—
—
—
—
—



hippocampoides



★Stylosanthes hispida
AJ320328
—
384 bp
455 bp
301 bp
372 bp 



Stylosanthes humilis
AJ320323
—
—
—
—
—



Stylosanthes ingrata
AJ320329
—
—
—
—
—



Stylosanthes leiocarpa
AJ320332
—
—
—
306 bp
71 bp



Stylosanthes linearifolia
AJ320367
—
—
—
—
—



Stylosanthes macrocarpa
AJ320369
—
—
—
—
—



Stylosanthes macrocephala
AJ320371
—
—
—
—
—



Stylosanthes macrosoma
AJ320333
—
—
—
—
—



Stylosanthes mexicana
AJ320374
—
—
—
—
—



Stylosanthes
AJ320336
—
—
—
—
—



montevidensis



Stylosanthes pilosa
AJ320377
—
—
—
—
—



Stylosanthes scabra
AJ320382
—
—
—
—
—



Stylosanthes seabrana
AJ320384
—
—
—
—
—



Stylosanthes sericeiceps
AJ320386
—
—
—
—
—



Stylosanthes subsericea
AJ320387
—
—
—
—
—



Stylosanthes sundaica
AJ320389
—
—
—
—
—



Stylosanthes sympodialis
AJ320391
—
—
—
—
—



Stylosanthes tomentosa
AJ320337
—
—
—
—
—



Stylosanthes tuberculata
AJ320392
—
—
—
—
—



★Stylosanthes viscosa
AJ320340
—
385 bp
149 bp
—
—






436 bp



Stylosanthes
AJ320316
—
—
—
—
—



hippocampoides



Stylosanthes humilis
AJ320327
—
—
—
301 bp
66 bp



Stylosanthes mexicana
AJ320373
—
—
—
—
—



Ormocarpum bernierianum
AF189036
—
—
—
—
—



Ormocarpum coeruleum
AF189037
—
—
—
—
—



Ormocarpum drakei
AF189039
—
—
—
—
90 bp
















TABLE 7D







Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Arachis
Ormocarpum flavum
AF189041
—
—
—
—
91 bp


Belonging to Leguminous
Ormocarpum keniense
AF068155
—
—
—
—
—



Ormocarpum kirkii
AF068152
—
—
—
—
—



Ormocarpum klainei
AF189044
—
—
—
—
91 bp



Ormocarpum
AF068154
—
—
—
—
91 bp



megalophyllum



Ormocarpum muricatum
AF068156
—
—
—
—
91 bp



Ormocarpum orientale
AF068159
—
—
—
—
91 bp



Ormocarpum pubescens
AF189045
—
—
—
—
91 bp



Ormocarpum rectangulare
AF189046
—
—
—
—
—



Ormocarpum schliebenii
AF189047
—
—
—
—
91 bp



Ormocarpum sennoides
AF068153
—
—
—
—
91 bp



Ormocarpum somalense
AF189048
—
—
—
—
—



Ormocarpum trachycarpum
AF189049
—
—
—
—
—



Ormocarpum trichocarpum
AF068158
—
—
—
—
—



Ormocarpum verrucosum
AF189050
—
—
—
—
91 bp



Chapmannia floridana
AF203543
—
—
462 bp
35 bp
226 bp 



Chapmannia prismatica
AJ320400
—
—
—
—
—



Chapmannia somalensis
AF203544
—
—
—
—
—



Chapmannia gracilis
AF203546
—
—
—
—
—



Chapmannia reghidensis
AF204232
—
—
—
—
—



Chapmannia sericea
AF203548
—
—
—
—
—



Chapmannia tinireana
AF203547
—
—
—
—
—



Ormocarpopsis aspera
AF068148
—
—
—
—
—



Ormocarpopsis calcicola
AF068145
—
—
—
—
—



Ormocarpopsis
AF068149
—
—
—
—
69 bp



itremoensis



Ormocarpopsis
AF068147
—
—
—
—
69 bp



mandrarensis



Ormocarpopsis parvifolia
AF068144
—
—
—
—
—



Ormocarpopsis
AF068146
—
—
—
—
69 bp



tulearensis



Diphysa humilis
AF068162
—
—
—
—
90 bp



Diphysa macrophylla
AF189029
—
—
—
—
—
















TABLE 7E







Peanut, SEQ NO: 18 & SEQ NO: 24 primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the
Diphysa suberosa
AF189034
—
—
—
—
90 bp


Genus Arachis Belonging
Fiebrigiella gracilis
AF203561
—
—
—
—
—


to leguminous
★Fissicalyx fendleri
AF189061
—
—
384 bp
349 bp
—







302 bp
340 bp








258 bp








150 bp








 66 bp


Related Species of the
Spigelia coelostylioides
AF177992
—
—
—
—
—


Genus Arachis Not
Spigelia hedyotidea
AF178005
—
—
—
—
—


Belonging to
Spigelia marilandica
AF177991
—
—
—
—
—


Leguminous









PCR described above was conducted using primers designed in the present invention. In this case, target 253 to 259 bp amplification products, 5 expected from the simulation results of the ITS-1 ̃5.8S rRNA gene ̃ITS-2 sequences of the genus Arachis, were obtained from 500 to 50 fg of peanut DNA. It is found from the results that even where 500 to 50 fg of peanut DNA is present, the peanut can be detected. In this connection, this sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity and specificity studied by PCR, it was confirmed that a wide range of plants in the genus Arachis including peanut were detectable using the present invention.
Example 6
A. Design of Oligonucleotide Primers for Detection of DNA from Wheat
(1) DNASequences of the Genus Triticum 
Regarding the genus Triticum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 29 DNA sequences registered in GenBank were examined to select suitable regions for the primer.

    1: Triticum aestivum (AF440679)
    2: Triticum aestivum (AF440676)
    3: Triticum aestivum (AF438191)
    4: Triticum aestivum (AF438188)
    5: Triticum aestivum (AF438187)
    6: Triticum aestivum (AF438186)
    7: Triticum baeoticum (AJ238901)
    8: Triticum urartu (AJ301803)
    9: Triticum turgidum subsp. dicoccum (AJ301801)
    10: Triticum monococcum (AJ301800)
    11: Triticum aestivum (AJ301799)
    12: Triticum monococcum (AJ245404)
    13: Triticum turgidum (AJ238919)
    14: Triticum turgidum (AJ238918)
    15: Triticum turgidum (AJ238917)
    16: Triticum turgidum (AJ238915)
    17: Triticum turgidum (AJ238913)
    18: Triticum turgidum (AJ238912)
    19: Triticum turgidum (AJ238911)
    20: Triticum timopheevii (AJ238924)
    21: Triticum timopheevii (AJ238923)
    22: Triticum timopheevii (AJ238922)
    23: Triticum timopheevii (AJ238921)
    24: Triticum timopheevii (AJ238920)
    25: Triticum turgidum (AJ238916)
    26: Triticum turgidum (AJ238914)
    27: Triticum urartu (AJ238902)
    28: Triticum aestivum (Z11761)
    29: Triticum monococcum (L11581)

(2) DNA Sequences of Other Common Allergenic Plants and Plants Widely Used for a Food Ingredient



The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: Buckwheat: Fagopyrum esculentum (AB000330)

(3) DNA Sequences of Rye, Barley and Oats



Regarding rye, barley and oats, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: Rye: Secale cereale (L36504)
    2: Barley: Hordeum vulgare (AF440678)
    3: Oat: Avena sativa (Z96893)

(4) DNA Sequences of Related Species of the genus Triticum 



As representatives of the DNA sequences of related species of the genus Triticum, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 70 DNA sequences registered in GenBank were selected. In this connection, the 70 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Triticum, each of which had the highest score in the corresponding genus other than genus Fagopyrum and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-2 sequence of wheat (Triticum aestivum Z11761).

    1: Ancestral species of wheat: Aegilops sharonensis (AF149195)
    2: Taeniatherum caput-medusae (L36505)
    3: Agropyron puberulum (L36482)
    4: Thinopyrum intermedium (AF507809)
    5: Lophopyrum elongatum (L36495)
    6: Pseudoroegneria spicata (L36502)
    7: Peridictyon sanctum (L36497)
    8: Australopyrum pectinatum (L36484)
    9: Amblyopyrum muticum (AF149202)
    10: Henrardia persica (L36491)
    11: Eremopyrum bonaepartis (L36490)
    12: Crithopsis delileana (L36487)
    13: Psathyrostachys fragilis (L36498)
    14: Heteranthelium piliferum (L36492)
    15: Critesion violaceum (L36488)
    16: Secale sylvestre (AJ409210)
    17: Haynaldia villosa (L36489)
    18: Bromus tectorum (L36485)
    19: Helictotrichon gervaisii (AJ389134)
    20: Festuca lasto (AF303418)
    21: Lagurus ovatus (AJ389166)
    22: Poa pratensis (AF171183)
    23: Pseudarrhenatherum longifolium (AJ389162)
    24: Alopecurus vaginatus (Z96921)
    25: Calamagrostis epigejos (AJ306448)
    26: Thisetum spicatum (AJ389168)
    27: Koeleria pyramidata (Z96911)
    28: Beckmannia eruciformis (AJ389164)
    29: Lolium persicum (AF171157)
    30: Diarrhena americana (AF019798)
    31: Arrhenatherum elatius (AF019795)
    32: Deschampsia christophersenii (AF486267)
    33: Piptochaetium fimbriatum (L36523)
    34: Vulpia fasciculata (AF303402)
    35: Phalaris truncata (L36522)
    36: Holcus lanatus (Z96919)
    37: Merxmuellera stricta (AF019871)
    38: Brachypodium mexicanum (AF019805)
    39: Austrostipa nodosa (AF019804)
    40: Ampelodesmos mauritanica (AF019799)
    41: Nassella viridula (L36521)
    42: Melica imperfecta (L36519)
    43: Achnatherum hymenoides (L36507)
    44: Austrodanthonia auriculata (AF367604)
    45: Notodanthonia laevis (AF019875)
    46: Oryzopsis exigua (AF019801)
    47: Chionochloa rigida (AF367597)
    48: Thysanolaena maxima (AF019854)
    49: Monachather paradoxus (A-F019852)
    50: Stipagrostis zeyheri (A-F019845)
    51: Arundo donax (AF019809)
    52: Zingeria biebersteiniana (AJ428836)
    53: Centothecalappacea (AF019814)
    54: Briza minor (L36510)
    55: Thibolium hispidum (AF367602)
    56: Rytidosperma pumilum (AF019878)
    57: Karroochloa purpurea (AF019874)
    58: Centropodia glauca (AF019861)
    59: Cortaderia archboldii (AF367620)
    60: Lamprothyrsus peruvianus (AF367605)
    61: Imperata cylindrica (A1F345653)
    62: Zizania latifolia (A-F169234)
    63: Prionanthium ecklonii (AF019866)
    64: Pentaschistis aspera (AFO 19865)
    65: Pentameris macrocalycina (A-FO19864)
    66: Molinia caerulea (AF019857)
    67: Dregeochloa pumilla (AFO 19853)
    68: Diplopogon setaceus (AF019851)
    69: Amphipogon amphopogonoides (AFO 19850)
    70: Aristida purpurea (AF019807)

(5) Oligonucleotide Primer Synthesis and Evaluation



Among ITS-2 sequences of the aforementioned 29 DNA sequences of the genus Triticum, there was determined nucleotide sequences which would specifically hybridize to all of the 29 DNA sequences of the genus Triticum through the study of the sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:28, 29 and 30. Subsequently, the oligonucleotide primers with SEQ NOs:28, 29 and 30 were synthesized.











Sense primer





5′-CGG CAT CTG GTC CCT CGT CT-3′
(SEQ NO:28)



 



Antisense primer



5′-GCG AGG ACG CCC ACC AT-3′
(SEQ NO:29)



 



5′GCA AAG ACG CCC ACC AT-3′
(SEQ NO:30)






B. PCR Simulation

Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 29 DNA sequences of the genus Triticum, the 8 DNA sequences of common allergenic plants other than wheat (buckwheat, peanut, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the DNA sequences of rye, barley and oat and the DNA sequences of Aegilops termed ancestral species of wheat and plants in related species of the genus Triticum used for breed improvement of wheat belonging to the tribe Triticeae. The simulation results are shown in Tables 8A to 8F. The meanings of symbols and numerical values in Tables 8A to 8F are explained below.

    ★: An obtained amplification product whose size almost matched the target 140 bp (±10 bp).
    W 2-6: Probability to obtain amplification products
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Triticum: 
    
        Sequences similar to the ITS-2 sequence of Triticum aestivum (Z11761) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them. Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Tables 8C-8F as the representative of the DNA sequences of related species of the genus Triticum. 

C. Preparation of Template DNA for PCR

(1) Samples Used for DNA Extraction

Wheat:

    
    


Commercially available seeds of wheat were used.
(2) DNA Isolation from Wheat
A DNA was isolated from wheat in the same way as in Example 1B (3). The isolated DNA preparation of wheat was diluted stepwise with sterilized ultrapure water to use as template DNA for PCR.
D. PCR
PCR was conducted in the substantially same way as Example 1C, except for use of the following primers and PCR program.
Primer:
The primer of SEQ NO:28 was used at 0.5 μM of final concentration and each primer of SEQ NOs:29 and 30 was used at 0.25 μM of final concentration.
PCR Program:
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 66° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 min.
The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA.
E. Results
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence of each plant. As a result, as shown in Tables 8A to 8F, it was predicted that target 93 to 95 bp amplification products would be obtained from the aforementioned 29 DNA sequences of the genus Triticum. On the other hand, it was predicted that no target 93 to 95 bp amplification product would be obtained from the 8 DNA sequences of common allergenic plants other than wheat (buckwheat, peanut, soybean, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown lice, pepper and mustard), the DNA sequences of rye, barley and oat, the 2 DNA sequences of related species of the genus Triticum belonging to the tribe Triticeae and the 51 DNA sequences of related species of the genus Triticum not belonging to the tribe Triticeae. In this connection, it was predicted by simulation that amplification products having almost 93 to 95 bp would be obtained from the DNA sequences of Aegilops termed ancestral species of wheat, plants in related species of the genus Triticum used for breed improvement of wheat belonging to the tribe Triticeae and some plants in related species of the genus Triticum not belonging to tribe Triticeae.







TABLE 8A







Wheat, SEQ No: 28 & SEQ NOs: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Triticum
★Triticum aestivum
AF440679
94 bp
—
335 bp
—
—







287 bp







218 bp



★Triticum aestivum
AF440676
94 bp
—
335 bp
—
—







287 bp







218 bp



★Triticum aestivum
AF438191
—
—
 95 bp
—
336 bp









288 bp









219 bp



★Triticum aestivum
AF438188
94 bp
—
335 bp
—
—







287 bp



★Triticum aestivum
AF438187
94 bp
—
335 bp
—
—







287 bp







218 bp



★Triticum aestivum
AF438186
94 bp
—
333 bp
—
—







285 bp







217 bp



★Triticum baeoticum
AJ238901
94 bp
—
—
—
—



★Triticum urartu
AJ301803
94 bp
—
334 bp
—
—







286 bp







217 bp



★Triticum turgidum subsp.
AJ301801
94 bp
—
335 bp
—
—



dicoccum



287 bp







218 bp



★Triticum monococcum
AJ301800
94 bp
—
286 bp
—
—







217 bp



★Triticum aestivum
AJ301799
94 bp
—
284 bp
—
—







215 bp



★Triticum monococcum
AJ245404
94 bp
—
—
—
—



★Triticum turgidum
AJ238919
94 bp
—
—
—
—



★Triticum turgidum
AJ238918
94 bp
—
—
—
—



★Triticum turgidum
AJ238917
94 bp
—
—
—
—



★Triticum turgidum
AJ238915
94 bp
—
—
—
—



★Triticum turgidum
AJ238913
94 bp
—
—
—
—
















TABLE 8B







Wheat, SEQ No: 28 & SEQ NOs: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Triticum
★Triticum turgidum
AJ238912
94 bp
—
—
—
—



★Triticum turgidum
AJ238911
94 bp
—
—
—
—



★Triticum timopheevii
AJ238924
94 bp
—
—
—
—



★Triticum timopheevii
AJ238923
94 bp
—
—
—
—



★Triticum timopheevii
AJ238922
93 bp
—
—
—
—



★Triticum timopheevii
AJ238921
94 bp
—
—
—
—



★Triticum timopheevii
AJ238920
94 bp
—
—
—
—



★Triticum turgidum
AJ238916
94 bp
—
—
—
—



★Triticum turgidum
AJ238914
94 bp
—
—
—
—



★Triticum urartu
AJ238902
94 bp
—
—
—
—



★Triticum aestivum
Z11761
94 bp
—
335 bp
—
—







287 bp







218 bp



★Triticum monococcum
L11581
94 bp
—
286 bp
—
—







217 bp


Common Allergenic Plants
Arachis hypogaea
AF156675
—
—
—
—
342 bp



(Peanut)





146 bp



Fagopyrum esculentum
AB000330
—
—
—
—
—



(Buckwheat)



Glycine max (Soybean)
U60551
—
—
—
—
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
215 bp



Malus x domestica
AF186484
—
—
—
—
—



(Apple)



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)


Plants Widely
Zea mays
U46648
—
—
—
—
—


Used for a Food
(Corn)


Ingredient
Oryza sativa (Brown rice)
AF169230
—
—
—
—
299 bp



Piper nigrum (Pepper)
AF275197
—
—
—
—
225 bp



Sinapis alba (Mustard)
X15915
—
—
—
—
—
















TABLE 8C







Wheat, SEQ No: 28 & SEQ NOs: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Rye, Barley
Secale cereale (rye)
L36504
—
—
—
—
—


and Oat
Hordeum vulgare (Barley)
AF440678
—
—
287 bp
—
—







218 bp



Avena sativa (Oat)
Z96893
—
—
—
—
—


Related Species of the Genus Triticum
★Aegilops sharonensis
AF149195
94 bp
—
335 bp
—
—


Belonging to Tribe Triticeae
(Ancestral species of wheat)



287 bp







218 bp



★Taeniatherum
L36505
94 bp
—
287 bp
—
—



caput-medusae



218 bp



★Agropyron puberulum
L36482
94 bp
—
287 bp
—
—







218 bp



★Thinopyrum intermedium
AF507809
94 bp
—
287 bp
—
—







218 bp



★Lophopyrum elongatum
L36495
94 bp
—
334 bp
—
—







286 bp







217 bp



★Pseudoroegneria spicata
L36502
94 bp
—
286 bp
—
—







217 bp



★Peridictyon sanctum
L36497
94 bp
—
286 bp
—
—







217 bp



★Australopyrum pectinatum
L36484
94 bp
—
286 bp
—
—







217 bp



★Amblyopyrum muticum
AF149202
94 bp
—
335 bp
—
—







287 bp







218 bp



★Henrardia persica
L36491
94 bp
—
334 bp
—
—







286 bp







217 bp



★Eremopyrum bonaepartis
L36490
94 bp
—
334 bp
—
—







286 bp







217 bp



★Crithopsis delileana
L36487
94 bp
—
335 bp
—
—







287 bp







218 bp
















TABLE 8D







Wheat, SEQ No: 28 & SEQ NOs: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus
★Psathyrostachys
L36498
94 bp
—
335 bp
—
—


Triticum Belonging to Tribe
fragilis



287 bp


Triticeae




218 bp



★Heteranthelium
L36492
94 bp
—
286 bp
—
—



piliferum



217 bp



Critesion violaceum
L36488
—
—
335 bp
218 bp
—







287 bp



Secale sylvestre
AJ409210
—
—
—
—
—


Related Species of the Genus Triticum
★Haynaldia villosa
L36489
94 bp
—
284 bp
—
—


Not Belonging to Tribe Triticeae




215 bp



★Bromus tectorum
L36485
94 bp
—
286 bp
—
—







217 bp



Helictotrichon
AJ389134
—
—
—
—
—



gervaisii



Festuca lasto
AF303418
—
—
—
—
—



★Lagurus ovatus
AJ389166
—
94 bp
—
—
—



Poa pratensis
AF171183
—
—
—
—
—



Pseudarrhenatherum
AJ389162
—
—
—
—
—



longifolium



Alopecurus vaginatus
Z96921
—
—
—
—
—



Calamagrostis epigejos
AJ306448
—
—
—
—
—



Trisetum spicatum
AJ389168
—
—
—
—
—



Koeleria pyramidata
Z96911
—
—
—
—
—



Beckmannia eruciformis
AJ389164
—
—
—
—
—



Lolium persicum
AF171157
—
—
—
—
—



Diarrhena americana
AF019798
—
—
—
—
—



Arrhenatherum elatius
AF019795
—
—
—
—
—



Deschampsia
AF486267
—
—
—
332 bp
—



christophersenii




215 bp



Piptochaetium
L36523
—
—
—
—
—



fimbriatum



Vulpia fasciculata
AF303402
—
—
—
—
—



Phalaris truncata
L36522
—
—
—
—
—



Holcus lanatus
Z96919
—
—
—
—
—
















TABLE 8E







Wheat, SEQ No: 28 & SEQ No: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Triticum
Merxmuellera stricta
AF019871
—
—
—
436 bp
—


Not Belonging to Tribe Triticeae





280 bp








211 bp



Brachypodium mexicanum
AF019805
—
—
—
—
—



Austrostipa nodosa
AF019804
—
—
—
—
—



Ampelodesmos
AF019799
—
—
—
—
—



mauritanica



Nassella viridula
L36521
—
—
—
—
—



Melica imperfecta
L36519
—
—
—
114 bp
—



Achnatherum hymenoides
L36507
—
—
—
286 bp
217 bp



Austrodanthonia auriculata
AF367604
—
—
—
282 bp
213 bp



Notodanthonia laevis
AF019875
—
—
—
283 bp
—



Oryzopsis exigua
AF019801
—
—
—
—
—



Chionochloa rigida
AF367597
—
—
—
281 bp
—








212 bp



Thysanolaena maxima
AF019854
—
—
—
—
—



Monachather paradoxus
AF019852
—
—
—
286 bp
217 bp









142 bp









 73 bp



Stipagrostis zeyheri
AF019845
—
—
—
—
—



Arundo donax
AF019809
—
—
—
289 bp
220 bp



Zingeria biebersteiniana
AJ428836
—
—
—
—
—



Centotheca lappacea
AF019814
—
—
—
—
—



Briza minor
L36510
—
—
—
—
—



Tribolium hispidum
AF367602
—
—
—
282 bp
213 bp



Rytidosperma pumilum
AF019878
—
—
—
282 bp
213 bp



Karroochloa purpurea
AF019874
—
—
—
282 bp
—








213 bp



Centropodia glauca
AF019861
—
—
—
281 bp
212 bp



Cortaderia archboldii
AF367620
—
—
—
280 bp
211 bp



Lamprothyrsus
AF367605
—
—
—
280 bp
211 bp



peruvianus



Imperata cylindrica
AF345653
—
—
—
—
—
















TABLE 8F







Wheat, SEQ No: 28 & SEQ No: 29 and 30 (two types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Related Species of the Genus Triticum
Zizania latifolia
AF169234
—
—
—
311 bp
—


Not Belonging to Tribe Triticeae





282 bp








213 bp



Prionanthium ecklonii
AF019866
—
—
—
471 bp
—








283 bp








214 bp



Pentaschistis aspera
AF019865
—
—
—
283 bp
—








214 bp



Pentameris macrocalycina
AF019864
—
—
—
283 bp
—








214 bp



Molinia caerulea
AF019857
—
—
—
—
—



Dregeochloa pumilla
AF019853
—
—
—
285 bp
—



Diplopogon setaceus
AF019851
—
—
—
285 bp
216 bp



Amphipogon amphopogonoides
AF019850
—
—
—
285 bp
216 bp



Aristida purpurea
AF019807
—
—
—
—
—









PCR described above was conducted using primers designed in the present invention. In this case, target 93 to 95 bp amplification products, expected from the simulation results of the ITS-2 sequences of the genus Triticum, were obtained from 500 to 50 fg of wheat DNA. It is found from the results that even where 500 to 50 fg of wheat DNA is present, the wheat can be detected. In this connection, this sensitivity correspond to a sensitivity wherein there can be detected 10 to 1 ppm of peanut DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity studied by PCR, it was confirmed that a wide range of the genus Triticum including wheat, ancestral species of wheat, and the majority of the tribe Triticeae were specifically detectable at a high sensitivity using the present invention.
Example 7
A. Design of Oligonucleotide Primers for Detection of DNA from Wheat
(1) DNA Sequences of the Genus Glycine 
Regarding the genus Glycine, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 50 DNA sequences registered in GenBank were examined to select suitable regions for the primer.

    1: Glycine max (U60551)
    2: Glycine max (L36612)
    3: Glycine max (AF144652)
    4: Glycine max (AF144651)
    5: Glycine max (BI674312)
    6: Glycine soja (U60550)
    7: Glycine soja (AF144653)
    8: Glycine soja (AJ009790)
    9: Glycine soja (AJ009791)
    10: Glycine soja (AJ224109)
    11: Glycine max (AJ011337)
    12: Glycine max (AJ009787)
    13: Glycine max (AF144654)
    14: Glycine cyrtoloba (U60548)
    15: Glycine tomentella (AF023447)
    16: Glycine tomentella (U60544)
    17: Glycine microphylla (U60537)
    18: Glycine tomentella (U60542)
    19: Glycine arenaria (U60543)
    20: Glycine tabacina (U60539)
    21: Glycine curvata (U60547)
    22: Glycine tomentella (AJ011345)
    23: Glycine pindanica (U60546)
    24: Glycine lactovirens (U60540)
    25: Glycine albicans (U60541)
    26: Glycine argyrea (U60535)
    27: Glycine tomentella (AF023446)
    28: Glycine latifolia (U60538)
    29: Glycine clandestina (U60534)
    30: Glycine tomentella (AF023445)
    31: Glycine dolichocarpa (AJ011340)
    32: Glycine dolichocarpa (AJ224110)
    33: Glycine canescens (AF023444)
    34: Glycine hirticaulis (U60545)
    35: Glycine tomentella (AJ011342)
    36: Glycine dolichocarpa (AJ011341)
    37: Glycine canescens (U60533)
    38: Glycine canescens (AJ011348)
    39: Glycine tabacina (AJ009788)
    40: Glycine tabacina (AJ009789)
    41: Glycine latrobeana (U60536)
    42: Glycine tomentella (AJ011344)
    43: Glycine tomentella (AJ011343)
    44: Glycine tomentella (AJ011338)
    45: Glycine tabacina (AJ011346)
    46: Glycine dolichocarpa (AJ011339)
    47: Glycine tabacina (AJ224111)
    48: Glycine falcata (U60549)
    49: Glycine latifolia (AJ009786)
    50: Glycine tabacina (AJ011347)

(2) DNA Sequences of Other Common Allergenic Plants and Plants Widely Used for a Food Ingredient



The DNA sequences described in Example 1A (2) “DNA Sequences of Other Common Allergenic Plants” and (3) “DNA Sequences of Plants Widely Used for a Food Ingredient” were selected. Regarding buckwheat, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: buckwheat: Fagopyrum esculentum (AB000330)

(3) DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient



The DNA sequences described in Example 3A (4) “DNA Sequences of Leguminous Plants Widely Used for a Food Ingredient” were selected. Regarding adzuki bean, ITS-2 sequences in the following DNA sequences registered in GenBank were selected.

    1: adzuki bean: Vigna angularis var. nipponensis (AB060088)

(4) DNA Sequences of Related Species of the Genus Glycine



As representatives of the DNA sequences of related species of the genus Glycine, 5.8S rRNA gene, ITS-1 and ITS-2 sequences in the following 5 DNA sequences registered in GenBank were selected. In this connection, the 5 DNA sequences were selected as representatives of the DNA sequences of related species of the genus Glycine, each of which had the highest score in the corresponding genus other than genus Glycine and a score of 60 bits or more among sequences of species belonging to the corresponding genus selected from sequences registrated in GenBank through a BLAST homology search using the ITS-2 sequence of soybean (Glycine max U60551).

    1: Ophrestia radicosa (AF467-484)
    2: Myrospermum sousanum (AF187086)
    3: Amphicarpaea bracteata (AF417019)
    4: Amphicarpaea edgeworthii (AF417013)
    5: Strophostyles umbellata (AFO69115)

(5) Oligonucleotide Primer Synthesis



Among ITS-2 sequences of the aforementioned 50 DNA sequences of the genus Glycine, there was determined nucleotide sequences which would specifically hybridize to all of the 50 DNA sequences of the genus Glycine through the study of the sequences. The thus determined nucleotide sequences are indicated as SEQ NOs:34 to 41. Subsequently, the oligonucleotide primers with SEQ NOs:34 to 41 were synthesized.









Sense primer:




5′-CTG ACC TCC CGC GAG CAC-3′
(SEQ NO:34)


 


Antisense primer:


5′-GCG TGG CTC ATC CAC CAT TTT ATC A-3′
(SEQ NO:35)


 


5′-GCG TTG CTC ATC CAC CAT TTT ATC A-3′
(SEQ NO:36)


 


5′-GCG TTG CTC ATC CAC CAT TTT GTC A-3′
(SEQ NO:37)


 


5′-GCA TTG CTC ATC CAC CAT TTT GTC A-3′
(SEQ NO:38)


 


5′-GCG CTG CTC ATC CGC CAT TTT GTC A-3′
(SEQ NO:39)


 


5′-GCG CTG CTC ATC CAC CAT TTT GTC A-3′
(SEQ NO:40)


 


5′-GCG TGG CTC ATC CAT TTT ATC A-3′
(SEQ NO:41)






B. PCR Simulation

Regarding the sense and antisense primer pairs, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels), which is the same as in Example 1 to examine whether target size of amplification products are obtained from the 50 DNA sequences of the genus Glycine, the 8 DNA sequences of common allergenic plants other than soybean (buckwheat, peanut, wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 5 DNA sequences of related species of the genus Glycine. The simulation results are shown in Tables 9A to 9C. The meanings of symbols and numerical values in Tables 9A to 9C are explained below.

    ★: An obtained amplification product whose size almost matched the target 87 to 89 bp (±10 bp).
    W 2-6: Probability to obtain amplification products 
    
        High Probability—W6>W5>W4>W3>W2—Low Probability
    
    
    Numerical values followed by bp:
    
        Each value was obtained by subtracting 2 from the value obtained in the simulation.
    
    
    (−): No amplification product was predicted.
    Related Species of the genus Arachis: 
    
        Sequences similar to the ITS-2 sequence of Glycine max (U60551) were searched by means of a BLAST homology search and the sequences having a score of 60 bits or more were selected from among them Each sequence having the highest score in each genus and having a score of 60 bits or more is shown in the following Table 9C as the representative of the DNA sequences of related species of the genus Glycine. 

C. Preparation of Template DNA for PCR

(1) Samples Used for DNA Extraction

Soybean:

    
    


Commercially available seeds of soybean were used.
(2) DNA Isolation from Soybean
A DNA was isolated from seeds of soybean in the same way as in Example 1B (3). The isolated DNA preparation of soybean was diluted stepwise with sterilized ultrapure water to use as template DNA for PCR.
D. PCR
PCR was conducted in the substantially same way as Example 1C, except for use of the following primers and PCR program.
Primer:
The primer of SEQ NO:34 was used at 0.5 μM of final concentration and each primer of SEQ NOs:36 and 37 was used at 0.25 μM of final concentration.
PCR Program:
PCR was conducted according to the following PCR program.
Pre-incubation at 95° C. for 15 min.; thereafter 45 cycles consisting of denaturation at 95° C. for 1 min., annealing at 68° C. for 1 min. and extension at 72° C. for 1 min.; followed by a final extension at 72° C. for 4 mm.
The quality of each of the template DNA used here was sufficient enough to be used for PCR based on the result of a separate PCR, in which target products were obtained using a primer pair to amplify a part of plant chloroplast DNA.
E. Results
Regarding the primers of the present invention, the simulation was conducted with PCR simulation software, Amplify 1.0 (Bill Engels) to examine the specificity to ITS-2 sequence of each plant. As a result, as shown in Tables 9A to 9C, it was predicted that target 87 to 89 bp amplification products would be obtained from the aforementioned 50 DNA sequences of the genus Glycine. On the other hand, it was predicted that no target 87 to 89 bp amplification product would be obtained from the 8 DNA sequences of other common allergenic plants (buckwheat, peanut, wheat, walnut, matsutake mushroom, peach, apple and orange), the 4 DNA sequences of plants widely used for a food ingredient (corn, brown rice, pepper and mustard), the 6 DNA sequence of leguminous plants widely used for a food ingredient (French bean, lima bean, lentil, chickpea, mung bean and adzuki bean) and the 3 DNA sequences of related species of the genus Glycine belonging to leguminous plants. In this connection, it was predicted by simulation that amplification products having almost 87 to 89 bp would be obtained from the DNA sequences of Amphicarpaea edgeworthii and Ophrestia radicosa, but the former can be identified by sequence analysis whether the genus Glycine or not and the latter may be able to be identified by appropriately using a commercially available ELISA kit of soybeans and various types of PCR and the like reported.







TABLE 9A







Soybean, SEQ NO: 34 & SEQ NOs: 35 to 41 (7 types) primer:


Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Glycine
★Glycine max (soybean)
U60551
89 bp
—
162 bp
—
—



★Glycine max (soybean)
L36612
89 bp
—
162 bp
—
—



★Glycine max (soybean)
AF144652
89 bp
—
162 bp
—
—



★Glycine max (soybean)
AF144651
89 bp
—
162 bp
—
—



★Glycine max (soybean)
BI674312
89 bp
—
162 bp
—
—



★Glycine soja
U60550
89 bp
—
162 bp
—
—



★Glycine soja
AF144653
89 bp
—
—
—
—



★Glycine soja
AJ009790
89 bp
—
161 bp
—
—



★Glycine soja
AJ009791
89 bp
—
160 bp
—
—



★Glycine soja
AJ224109
89 bp
—
160 bp
—
—



★Glycine max (soybean)
AJ011337
89 bp
—
160 bp
—
—



★Glycine max (soybean)
AJ009787
89 bp
—
160 bp
—
—



★Glycine max (soybean)
AF144654
87 bp
—
155 bp
—
—



★Glycine cyrtoloba
U60548
89 bp
—
162 bp
—
—



★Glycine tomentella
AF023447
89 bp
—
162 bp
—
—



★Glycine tomentella
U60544
89 bp
—
162 bp
—
—



★Glycine microphylla
U60537
89 bp
—
162 bp
—
—



★Glycine tomentella
U60542
89 bp
—
162 bp
—
—



★Glycine arenaria
U60543
89 bp
—
162 bp
—
—



★Glycine tabacina
U60539
89 bp
—
357 bp
—
—







162 bp



★Glycine curvata
U60547
89 bp
—
162 bp
—
—



★Glycine tomentella
AJ011345
89 bp
—
161 bp
—
—



★Glycine pindanica
U60546
89 bp
—
162 bp
—
—



★Glycine lactovirens
U60540
89 bp
—
162 bp
—
—



★Glycine albicans
U60541
89 bp
—
162 bp
—
—



★Glycine argyrea
U60535
89 bp
—
162 bp
—
—



★Glycine tomentella
AF023446
89 bp
—
162 bp
—
—



★Glycine latifolia
U60538
89 bp
—
357 bp,
—
—







162 bp



★Glycine clandestina
U60534
89 bp
—
162 bp
—
—



★Glycine tomentella
AF023445
89 bp
—
162 bp
—
—



★Glycine dolichocarpa
AJ011340
89 bp
—
161 bp
—
—
















TABLE 9B







Soybean, SEQ NO: 34 & SEQ NOs: 35 to 41 (7 types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Genus Glycine
Glycine dolichocarpa
AJ224110
89 bp
—
161 bp
—
—



★Glycine canescens
AF023444
89 bp
—
161 bp
—
—



★Glycine hirticaulis
U60545
89 bp
—
162 bp
—
—



★Glycine tomentella
AJ011342
89 bp
—
161 bp
—
—



★Glycine dolichocarpa
AJ011341
89 bp
—
161 bp
—
—



★Glycine canescens
U60533
89 bp
—
162 bp
—
—



★Glycine canescens
AJ011348
89 bp
—
162 bp
—
—



★Glycine tabacina
AJ009788
89 bp
—
160 bp
—
—



★Glycine tabacina
AJ009789
89 bp
—
161 bp
—
—



★Glycine latrobeana
U60536
89 bp
—
162 bp
—
—



★Glycine tomentella
AJ011344
89 bp
—
—
—
—



★Glycine tomentella
AJ011343
89 bp
—
—
—
—



★Glycine tomentella
AJ011338
89 bp
—
—
—
—



★Glycine tabacina
AJ011346
89 bp
—
161 bp
—
—



★Glycine dolichocarpa
AJ011339
89 bp
—
—
—
—



★Glycine tabacina
AJ224111
89 bp
—
161 bp
—
—



★Glycine falcata
U60549
89 bp
—
439 bp
—
—







184 bp



★Glycine latifolia
AJ009786
89 bp
—
357 bp
—
—



★Glycine tabacina
AJ011347
89 bp
—
161 bp
—
—
















TABLE 9C







Soybean, SEQ NO: 34 & SEQ NOs: 35 to 41 (7 types) primer: Amplification products














GenBank







Scientific Name
Accession


(Common Name)
No.
W6
W5
W4
W3
W2

















Other Common Allergenic Plants
Arachis hypogaea (Peanut)
AF156675
—
—
—
—
—



Fagopyrum esculentum
AB000330
—
—
—
—
—



(Buckwheat)



Triticum aestivum (Wheat)
AJ301799
—
—
—
—
—



Juglans regia (Walnut)
AF303809
—
—
—
—
—



Tricholoma matsutake
U62964
—
—
—
—
—



(Matsutake mushroom)



Prunus persica (Peach)
AF185621
—
—
—
—
—



Malus x domestica
AF186484
—
—
—
119 bp
387 bp



(Apple)





137 bp









121 bp









107 bp









105 bp



Citrus sp.
E08821
—
—
—
—
—



(Valencia orange)


Plants Widely
Zea mays (Corn)
U46648
—
—
—
—
—


Used for a
Oryza sativa (Brown rice)
AF169230
—
—
—
—
217 bp


Food
Piper nigrum (Pepper)
AF275197
—
—
—
—
216 bp


Ingredient
Sinapis alba
X15915
—
—
—
—
—



(Mustard)


Leguminous Plants Widely
Phaseolus vulgaris
AF115169
—
—
—
—
376 bp


Used for a Food Ingredient
(French bean)



Phaseolus lunatus
AF115175
—
—
—
—
—



(Lima bean)



Lens culinaris subsp.
AF228066
—
—
—
—
—



culinaris (Lentil)



Cicer arietinum (Chickpea)
AJ237698
—
—
—
—
—



Vigna radiata (Mung bean)
X14337
—
—
—
—
—



Vigna angularis var.
AB060088
—
—
—
—
—



nipponensis (Adzuki bean)


Related Species of
★Ophrestia radicosa
AF467484
89 bp
—
162 bp
—
—


the Genus Glycine
Myrospermum sousanum
AF187086
—
—
161 bp
—
—


Belonging to
Amphicarpaea bracteata
AF417019
—
—
—
—
—


Leguminous Plants
★Amphicarpaea edgeworthii
AF417013
—
88 bp
—
—
—



(Wild bean)



Strophostyles umbellata
AF069115
—
—
—
—
—









PCR described above was conducted using primers designed in the present invention. In this case, target 87 to 89 bp amplification products, expected from the simulation results of the ITS-2 sequences of the genus Glycine, were obtained from 500 to 50 fg of soybean DNA. It is found from the results that even where 500 to 50 fg of soybean DNA is present, the soybean can be detected. In this connection, this sensitivity corresponds to a sensitivity wherein there can be detected 10 to 1 ppm of soybean DNA contained in the sample DNA when PCR was conducted with, as a template, 50 ng of DNA isolated from some samples.
Consequently, in conjunction with the results of specificity studied by PCR simulation, and the results of sensitivity studied by PCR, it was confirmed that a wide range of the genus Glycine including soybean were specifically detectable at a high sensitivity using the present invention.

## Claims

1. A method for detecting species in the genus Glycine in cases where even one species of the genus Glycine is contained in a subject to which the method is to be applied such as a food ingredient or product, which comprises the steps of:
a) collecting 45S rRNA precursor gene sequences containing an ITS sequence of the genus Glycine and plants thought to be related species thereof and then selecting the region common to the genus Glycine in the 45S rRNA precursor gene sequences;
b) selecting the base specific to the genus Glycine from the region;
c) designing primers (A) and/or (B) having the specific base at the 3′ end thereof, which primers can hybridize under the stringent conditions to a nucleic acid molecule having a common nucleotide base sequence for all species in the genus Glycine in the 45S rRNA precursor gene sequence thereof, wherein the 3′ end of primer (A) can complementarily bind to a base in the ITS-1 Sequence of the genus Glycine when the primer hybridizes to the nucleic acid molecule while the 3′ end of primer (B) can complementarily bind to a base in the ITS-2 sequence of the genus Glycine when the primer hybridizes to the nucleic acid molecule;
d) isolating DNA from the subject to which the method is to be applied;
e) conducting PCR amplification for the isolated DNA using at least one member selected from the group consisting of the primers (A) and (B); and
f) identifying the presence of the resulting amplification product from PCR containing at least a part of ITS-1 or ITS-2 sequence of the genus Glycine.

2. The method of claim 1, wherein the nucleic acid molecule having a common nucleotide sequence for all species in the genus Glycine in 45S rRNA precursor gene sequence thereof is a nucleic acid molecule having a common specific nucleotide sequence for all species in the genus Glycine in l-ITS-1 sequence thereof.

3. The method of claim 1, wherein the nucleic acid molecule having a common nucleotide sequence for all species in the genus Glycine in 45S rRNA precursor gene sequence thereof is a nucleic acid molecule having a common specific nucleotide sequence for all species in the genus Glycine in ITS-2 sequence thereof.

4. The method of claim 2, wherein the step of conducting PCR comprises using the primers (A) and (C), which primer (C) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which ITS-1, 5.8S rRNA gene, ITS-2 and LSU rRNA gene sequences of the genus Glycine are continuously bonded.

5. The method of claim 3, wherein the step of conducting PCR comprises using the primers (B) and (D), which primer (D) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which SSU rRNA gene, ITS-1, 5.8S rRNA gene and ITS-2 sequences of the genus Glycine are continuously bonded.

6. The method of claim 2, wherein the step of conducting PCR comprises using the primers (A) and (E), which primer (E) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which SSU rRNA gene and ITS-I sequences of the genus Glycine are continuously bonded.

7. The method of claim 3, wherein the step of conducing PCT comprises using the primers (B) and (F), which primer (F) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in which ITS-2 and LSU rRNA sequences of the genus Glycine are continuously bonded.

8. The method of claim 3, wherein the common specific nucleotide sequence for all species in the genus Glycine in ITS-2 sequence thereof is selected from the group consisting of SEQ NOs:31, 32 and 33, and complementary nucleotide sequences thereof.

9. The method of claim 1, wherein the primer (B) is selected from the group consisting of SEQ NOs:34, 35, 36, 37, 38, 39, 40 and 41.

10. The method of claim 4, wherein the primer (C) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in 5.8S rRNA gene sequence of the genus Glycine, and 3′ end of primer (C) can complementarily bind to a base in 5.8S rRNA gene sequence of the genus Glycine when the primer hybridizes to the nucleic acid molecule.

11. The method of claim 5, wherein the primer (D) can hybridize under stringent conditions to a nucleic acid molecule having a part of a nucleotide sequence in 5.8S rRNA gene sequence of the genus Glycine, and 3′ end of primer (D) can complementarily bind to a base in 5.8S rRNA gene sequence of the genus Glycine when the primer hybridizes to the nucleic acid molecule.

12. The method of claim 10, wherein the part of nucleotide sequence in 5.8S rRNA gene sequence of the genus Glycine is selected from the group consisting of SEQ NO:1 and a complementary nucleotide sequence thereof.

13. The method of claim 11, wherein the part of nucleotide sequence in 5.8S rRNA gene sequence of the genus Glycine is selected from the group consisting of SEQ NO:1 and a complementary nucleotide sequence thereof.

14. The method of claim 4, wherein the primer (C) is selected from the group consisting of SEQ NOs:2, 3 and 4.

15. The method of claim 5, wherein the primer (D) is selected from the group consisting of SEQ NOs:5, 6 and 7.

16. The method of claim 1, wherein the step of conducting PCR comprises using a combination of a primer of SEQ NO:28 and a primer selected from the group consisting of SEQ NOs:35, 36, 37, 38, 39, 40 and 41 as the primer (B).