# Patent

## Title
Genetic identification and validation of Echinacea species

## Abstract

A method for identification and validation of Echinacea is disclosed. Primers are designed based on information analysis of sequences from a large number of Echinacea species to amplify certain segments of genomic DNA to identify the species. Primers and methods are also disclosed to amplify other plant species that are frequently found in adulterated herbal samples of Echinacea.

## Background
RELATED APPLICATIONS
This application claims priority of U.S. Provisional Application No. 60/908,586, filed Mar. 28, 2007, the content of which is hereby incorporated into this application by reference.


SEQUENCE LISTING
This application is accompanied by a sequence listing that accurately reproduces the sequences described herein.
BACKGROUND
1. Field of the Invention
The present disclosure pertains to methods for identification of plant species using molecular genetic tools. More particularly, the disclosure relates to validation of Echinacea species through DNA polymerase chain reaction (PCR) using primers designed based on their information content.
2. Description of Related Art
The use of dietary supplements, particularly vitamins and botanicals, have become very popular in the United States and Europe. A recent survey conducted by the FDA found that 16 million Americans use botanical supplements. The industry has consistently grown at a rate of approximately 25% annually since 1990. Supplements containing the Purple Coneflower, Echinacea purpurea and other related species, is one of the top selling products. A survey published in USA Today indicated that 19% of adult Americans have used Echinacea for the treatment of colds and flu symptoms. The plant contains substances that have been shown to non-specifically stimulate the immune system in animals and man (Stimpel et al. 1984; Roesler et al. 1991a, 199b). Bioactive high-molecular-weight polysaccharides are believed to be involved in a stimulation of lymphocytic macrophages and B cells with the production of various cytokines (e.g. interleukins, tumor necrosis factor and interferons). The response has been measured in-vivo and verified in clinical studies (Braunig et al 1992; Schoneberger et al. 1992). These plant used in its typical dosage forms has also been shown to non-toxic with no measurable side effects or known contraindications (Mengs et al. 1991).
The North American genus Echinacea consists of nine species and multiple varieties and has a distribution centering on Arkansas, Oklahoma, Missouri and Kansas. While the species are relatively distinct from one another, specific differences are narrowly defined. All taxa will hybridize when brought together and considerable natural hybridization occurs (McGregor 1968). E. angustifolia and E. atrorubens, which show geographic introgression, exhibit considerable overlap in stem and petiole structures. Hybrids of E. simulata and E. sanguinea appear to be very similar to the Arkansas race of E. pallida, and E. simulata has in the past been considered a variety of E. pallida. Further studies revealed differences in pollen size and morphology and that pallida was polyploid, and those natural hybrids were triploid and sterile. Because of hybridization, similarities between species and overlapping ranges, the identification of Echinacea species based on morphological characteristics alone is a complex process that is prone to ambiguity.
The currently practiced methods for identification of botanical species from processed plant materials (dried roots, stems, leaves, flower and seed) involve the chemical identification of chromatographic profiles of plant secondary products. These chromatographic techniques include thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). The identification of a plant species is accomplished by comparison of chemical constituent profiles with chemical standard mixtures derived from extracts of a voucher plant specimen. Confirmations are often made by microscopic evaluation of the processed plant material. The results obtained from such analysis are subject error and misinterpretation because many plant secondary products are common within families and genera, and their natural variability can be influenced by growth cycle and habitat.
Public safety and product efficacy demand uniform state-of-the-art quality assurance programs. The Food & Drug Administration has a mandate to begin regulation of the dietary supplement industry for quality assurance and good manufacturing practices (US Code, 63 Fed. Reg. 23624). With the implementation of regulations to ensure quality control of product manufacture of phytomedicinals (21 CFR 201.128, Food & Drug Cosmetic Act), species certification is likely to be required of all botanical raw materials. Even if not required by the FDA, such certification increases consumer confidence and provides a competitive market advantage for producers of the certified product. It is also conceivable that, certification may become contractually mandated by international importers of phytomedicinals or by traders in commodity futures markets.
Molecular genetic analysis of highly conserved genes have been used in species identification because distinct genetic sequences are found in different species and in some instances individuals within the same species. These profiles can be determined with great precision and when properly applied are reproducible, rapid and scalable to large numbers of specimens. In addition to DNA sequence analysis, various DNA-based methods have been used to identify differences among individuals, populations and plant or animal species. In plant taxonomy, the techniques of RFLP (Restriction Fragment Length Polymorphism) and AFLP (Amplified Fragment Length Polymorphisms) have been used to characterize genetic differences between species and their sub-populations. Both procedures generate an array of DNA fragments of varying lengths is generated by these procedures that are unique for a given individual organism. The RFLP method is does not provide optimal discrimination of species (Hollis et al. 1999), requires highly purified DNA, and is not easily scaled to large numbers of specimens and can give erroneous results due to epigenetic differences between species (Jupe and Zimmer 1990). AFLP has been used to characterize the taxonomy of the genus of Echinacea (Baum et al. 2002). This technique, however, can be difficult to reproduce in different laboratories. This may be due to the fact that the AFLPs are derived from highly repetitive sequences that are not stable in plant genomes (Reamon-Buttner S M et al. 1999).
SUMMARY
It is hereby disclosed a genetic method for the identification of each of the nine species of commercially-produced Echinacea. It is an objective of this invention to provide a reliable method to certify the purity of the plant material distributed in the US and overseas. Accurate identification of each species would detect adulteration with other species and be impervious to adulteration with active chemical constituents. It is another goal of this invention to develop genetic technology that will be commercialized into both products (kits) and analytical services for distributors of botanical products, neutraceutical manufacturers and agricultural producers. The use of DNA sequence analysis to analyze ingredients in herbal supplements for the identification of species represent a significant improvement over existing methodology in Echinacea taxonomy. Existing HPLC methods for evaluating Echinacea extracts cost $200-300 per specimen and are not as accurate as the methodology present disclosed. In addition to its accuracy, the methods disclosed here are also simple to perform with a cost similar to or less than HPLC. High throughput (multiwell plate) testing may be performed at a cost less than current HPLC methods and provide more information in each test. In manufacturing a typical neutraceutical product, the cost of quality control of raw materials is estimated to be approximately 1% of product sales. If this DNA analytical procedure is applied to 20% of QC costs for all botanical supplement products, then the potential for sales of this analytical service is  ̃$10 million annual sales in today's market.
The methods hereby disclosed are based upon the technology disclosed in U.S. Pat. No. 5,849,492 (the '492 patent), which describes DNA amplification and sequencing assay (omnibus PCR) to accurately identify a wide spectrum of organisms in vitro without culturing or growing the organisms. The '492 patent also describes primer sequences for 16S rDNA and 28S rDNA for identification of prokaryotic and eukaryotic organisms, respectively. The teachings of the '492 patent are hereby expressly incorporated into this disclosure by reference.
The sequences of ribosomal RNA molecules have been used to identify genetic differences between bacterial species (Woese, 1987). Other DNA technologies, such as Amplified Restriction Fragment Polymorphism analysis, have produced ambiguous results when used to identify species (See, e.g., Binns et al. 2002; Kim et al 2004; Bobowski et al., 1999). The present work is aimed at developing a method to use DNA from the coding sequences of ribosomal RNA to distinguish different Echinacea species. Briefly, ribosomal nucleotide sequences that are either published or unpublished are used to investigate their suitability in distinguishing multiple varieties of five different Echinacea species and Parthenium integrifolium, a reported adulterant in Echinacea products (Bauer, 1998b). These studies may also reveal other intraspecies genetic variation that can distinguish different varieties.
Thus, it is disclosed herein a number of oligonucleotide pairs for amplification of ribosomal DNA from a plant material, including primer set 1, primer set 2, primer set 3, and primer set 4, the sequences of which are detailed in the Examples. It is also disclosed methods for identifying a plant species or for validating a plant material based on the primary structure of the DNA contained within the plant species or the plant material, said method comprising amplification of a segment of the internal transcribed spacer region of the ribosomal DNA of said plant species. In one aspect, the primer pair used for the amplification is Echinacea specific, or in other words, the primers specifically amplify DNA from Echinacea species but do not amplify DNA from Parthenium species. In another aspect, the primer pair used for the amplification is Parthenium specific, namely, they specifically amplify DNA from Parthenium species but do not amplify DNA from Echinacea species. Preferably, the primer pair used for the amplification is selected from the group consisting of primer set 1, primer set 2, primer set 3, and primer set 4.
It is also disclosed a method for determining the existence of an organism or its derivatives in a material. For purpose of this disclosure, derivatives of an organism means a material that is derived from any parts or tissues of the organism. In one embodiment, the method may include the following steps: (1) a first DNA amplification may be performed to amplify a segment on a ribosomal DNA, wherein a first primer pair is used as PCR primer. DNA from at least one species belonging to the same genus as said organism may be used as template for the first DNA amplification; (2) the resultant PCR product resulting from the first amplification may be sequenced; and (3) a second DNA amplification may be performed using as PCR primer a second primer pair and using as template DNA prepared from said material.
In one aspect, the selection of the first primer pair may include the steps of: (a) searching for a divergent segment of the DNA from said at least one species with low average information content determined quantitatively surrounded by two conserved segments of said DNA with high average information content determined quantitatively; and (b) designing the first primer pair for PCR amplification of said divergent segment by constructing a sequence logo for said DNA such that said primers contain a set of sequences present in said sequence logo that encompass the nucleotide variability of said conserved segments, said primer pair being able to anneal to said conserved segments for amplication of said divergent segment. The calculation of average information content and the construction of logo may be performed based on methods taught in the '492 patent.
The second primer pair may be selected by first searching the sequences obtained from step (2) above for at least one segment of DNA with significant interspecies variations; and then by designing the second primer pair for DNA amplification, wherein said second primer pair comprises at least one interspecies variation. More preferably, the second primer pair includes at least one interspecies variation such that it only amplifies DNA from certain species, but not others. Most preferably, the second primer pairs are selected such that one primer pair would amplify most species of either the Echinacea or Parthenium genus, but not both.
In one embodiment, the template for the amplification is DNA prepared from a target sample, such as target sample obtained from harvest of a previous growing season, which typically means a growing season at least 6 months before. Thus, the target samples may be stored for 6 months, or even longer.
In another aspect of the present disclosure, the target sample may contain specimen from more than one species, such as, for example, from at least two species selected from the group consisting of Echinacea, Parthineum and Rudbeckia. Preferably, the target sample contains specimen from Echinacea and Parthineum. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows multiple alignment of published ribosomal ITS1 sequences from several Echinacea species, E. paradoxa (SEQ ID No. 1), E. simulata (SEQ ID No. 2), E. pallida (SEQ ID No. 3), E. purpurea (SEQ ID No. 4), E. tenesseensis (SEQ ID No. 5), E. atrorubens (SEQ ID No. 6).
FIG. 2 shows neighbor-joining dendrograms depicting phylogenetic relationships of Echinacea species based on corresponding sequence differences in the ribosomal intergenic spacer region. (A) ITS1 (B) ITS2.
FIG. 3 is a DNA sequence alignment of a segment of the ITS1 region obtained by amplification and sequencing of the ITS1 regions from genomic DNA extracted from Echinacea specimens collected as described in this disclosure, with the 11 sequences shown designated as SEQ ID Nos. 7-17, respectively.
FIG. 4 is a DNA sequence alignment of a segment of the ITS1 region obtained by amplification and sequencing of the ITS1 regions from genomic DNA extracted from Parthenium specimens collected as described in this disclosure, with the 4 sequences shown designated as SEQ ID Nos. 18-21, respectively.
FIG. 5 is a DNA sequence alignment of a segment of the ITS2 region obtained by amplification and sequencing of the ITS2 regions from genomic DNA extracted from various E. angustifolia specimens collected as described in this disclosure, with the 3 sequences shown designated as SEQ ID Nos. 22-24, respectively.
FIG. 6 is a DNA sequence alignment of a segment of the ITS2 region obtained by amplification and sequencing of the ITS1 regions from genomic DNA extracted from Echinacea and Parthenium specimens collected as described in this disclosure, with the 57 sequences shown designated as SEQ ID Nos. 25-81, respectively.
FIG. 7 shows that the Echinacea specific primer sets 3 and 4 generate PCR products only when Echinacea DNA is present as template, while Parthenium specific primers, sets 1 and 2, only amplify Parthenium genomic DNA.
FIG. 8 shows the effects of the source of DNA and the methods of purifying the template DNA on the PCR amplification.
FIG. 9 shows results of agarose gel electrophoresis of amplified internal transcribed spacer sequences from several Echinacea species under different concentrations of Mg2+.



DETAILED DESCRIPTION
Polymerase chain reaction (PCR) used in this disclosure may be performed as described by Mullis and Faloona (1987) and any subsequent modification to the methodology originally invented by Mullis. All temperatures stated in this disclosure are centigrade unless otherwise specified.
Since almost all organisms employ ribosomes to synthesize proteins, ribosomal subunits have been structurally and functionally conserved throughout the eons. As a result, ribosomal RNAs from widely differing species may differ in only a small number of nucleotides. These limited sequence variations may be used to characterize the evolutionary or phylogenetic relationships between the organisms and to identify a specific organism. Briefly, information (in bits) may be used to precisely quantify both the similarities and divergence among 16S gene sequences, because information measures the number of choices between two equally likely possibilities (Schneider et al., J. Mol. Biol. 188: 415-431, 1986). Variable positions in a multiply aligned set of 16S rDNA sequences approach zero bits and homologous or highly conserved sequences have nearly two bits in a sequence logo (Stephens & Schneider, Nucl. Acids Res. 18: 6097-6100, 1990), which displays the average information content (Rsequence) and frequencies of each nucleotide at each position.
The average information in bits of a related set of sequences, Rsequence, represents the total sequence conservation:




  
    
      
        
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f(b,l) is the frequency of each base b at position l,

e(n(l)) is a correction for the small sample size n at position l.

A sequence logo may then be constructed based on the Rsequence to locate segments consisting of sequences with low information content flanked on either side by sequences with high information content.
Both the similarities and differences between organisms may be used to obtain the identity of these organisms. Morphological and biochemical properties have been used to differentiate among different organisms. However, these methods can be time consuming and can be inaccurate. False results may be generated if the culturing condition used is less than optimal, and thus the assay fails to identify the organism that is present in a specimen. According to the present disclosure, nucleic acid sequences of homologous genes in different species may reveal the identity of infectious agents. The frequency and arrangement of nucleotide differences indicate the degree to which two organisms have diverged from a common ancestor. In a preferred embodiment, the sequences of ribosomal DNA may be used to identify genetic differences between different species.
In order to ensure that the widest spectrum of organisms may be identified, it is desirable to apply the information theory-based sequence analysis to a greatest possible number of species to select for sequences in the homologous 16S ribosomal RNA genes (16S rDNA) for DNA amplification. In one embodiment, full length 16S rDNA sequences from a set of bacterial species (2184 organisms obtained from Genbank v88; National Library of Medicine) having the broadest possible taxonomic distribution are used to design amplification experiments (Saiki et al., Science 230: 1350-1354, 1985).
The total information content at each position may be used as the basis for selecting phylogenetically-informative regions flanked by segments about 18 bp or more showing sufficient sequence conservation to be used as primers for the PCR amplification reaction. The ratio of the number of bits of each nucleotide at each position to the total number of bits at that site may determine the proportion of a particular nucleotide at degenerate sites in the oligonucleotide primer. A ratio of 0.001 may be taken as the minimum proportion required to include this nucleotide in a degenerate site (see below). Otherwise, the primer may be designed to be homogeneous at that position.
A sequence logo may be used to locate several segments consisting of sequences with low information (>100 nucleotides, average Rsequence=0.2) content flanked on either side by sequences with high information content and tested experimentally (Rogan et al., 1995; Tooley P W, Salvo J J, Schneider T D, Rogan P K: Phylogenetic inference using information theory-based PCR amplification. J Phytopathology, 146(8-9): 427-430, 1998; U.S. Pat. No. 5,849,492). Alternatively, alignment and visual inspection of sequences from different species may be employed to select a set of primers suitable for PCR amplification of segments of DNA from these species. An ideal set of primers preferably anneals to regions that show relatively high sequence similarity across species, and also preferably flanks a segment of DNA that demonstrates high sequence variation among species.
The PCR product may be purified using a number of established methods for DNA purification. Preferably, the PCR product is purified using magnetic separation, or gel purification. Magnetic separation may have a higher yield of recovered PCR product than gel purification. At least one primer may be biotinylated if magnetic separation of PCR product is to be used. Although gel purification produces relatively lower yield and requires higher amount of the amplified DNA, gel purification has proven helpful in some instances in reducing the problem of concatomers of PCR products that may cause difficulty in obtaining clean sequence data. One potential drawback for gel purification is that it may not be as conducive to automation as magnetic separation. Magnetic separation is the preferred method for purifying the PCR products.
The purified PCR products may be characterized by sequencing or other molecular tools. Sequencing methods such as the dideoxy method or the chemical method may be used Sanger F, Nicklen S, Coulson A R, “DNA sequencing with chain-terminating inhibitors.” Proc Natl Acad Sci USA. 1977 74 (12):5463-7; Maxam A M and Gilbert W, “Sequencing end-labeled DNA with base-specific chemical cleavages.” Methods Enzymol. 1980; 65(1):499-560. The sequences may be read from a film exposed to the sequencing gel or may be obtained using an automated sequencing machine. Other molecular tools such as mass spectrometry or other structural analysis tools capable of discerning the difference in primary sequence may also be used to characterize the PCR products. Some of these tools for molecular characterization of DNA have been summarized in a review article by V. K. Khanna, “Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: A multidisciplinary development unifying molecular biology, chemical and electronics engineering” Biotechnology Advances, 2007, 25:85-98, which is hereby incorporated by reference.
If conventional DNA sequencing is used, when the sequencing run is finished, DNA sequence analysis software, such as Visible Genetics OpenGene, may be used to align and base call the electropherogram. Preferably, the sequence data are manually checked and edited to obtain as clean and accurate a sequence as possible prior to sequence analysis. In some situations, the electropherogram may need to be manually aligned and/or base called because of the limitations of the software.
Using the PCR and sequencing methodology described above, there may be instances where the sequence is not readable because there are multiple peaks at several locations in the sequence. This problem typically arises when more than one organisms are present in the same sample. To eliminate this problem, a constant denaturing gel electrophoresis (CDGE) protocol may be used, which allows DNA to be separated on the basis of sequence composition and duplex stability in a vertical polyacrylamide gel.
Phylogenetic relationships have been inferred from nucleotide sequence variations in the internal transcribed spacer regions (ITS) of 18S-25S nuclear ribosomal DNA genes of angiosperms. The taxonomies from ITS data, which are generally concordant with phylogenies derived from chloroplast and other nuclear gene sequences, have resulted in some reclassification of angiosperms at the ordinal level (Soltis and Soltis 2000). With few exceptions (e.g. Francisco-Ortega et al. 1999), different angiosperm species are resolved by ITS sequence analysis, though the tree topologies can differ from those based on vegetative and floral morphology (e.g. Aceto et al. 1999). ITS-based taxonomies are congruent with other nuclear gene phylogenies (e.g. Emshwiller and Doyle 1999), but nevertheless, can display higher bootstrap values than those based on chloroplast genes (e.g. Gielly et al. 1996), due to higher levels of interspecies sequence divergence in the ITS region.
The ribosomal intergenic spacer sequences (ITS) can be used to differentiate among several Echinacea species. Two different sequence regions, known as ITS1 and ITS2, have been shown to exhibit the largest amount of interspecies variability. Six sequences of the ITS1 region from different Echinacea species have been reported in GenBank (Accession nos. U73148, U73149, U73150, U73151, U73152, U73153). These sequences have not been peer reviewed but may be used to demonstrate their suitability for use in differentiating between different Echinacea species.
As shown in FIG. 1, variations in nucleotide sequence may be found in each ITS1 DNA sequence from different Echinacea species. Bolded positions in the alignment illustrate sites where these species may be distinguished based on sequence variations. For instance, E. tenesseensis lacks cytosine at position 10 in the multiple alignment shown in FIG. 1, whereas it is present in the other species. Similarly, E. atrorubens differs at positions 42 and 50 of the alignment. E. purpurea and E. tenesseensis both contain adenosine at position 66, whereas guanosine is present in the other species. Only E. paradoxa and E. simulata lack guanosine at position 162, and may be distinguished from each other based on an extra adenosine in E. simulata at position 220 in the multiple alignment shown in FIG. 1.
Certain intraspecies variations based on ITS polymorphism may exist in species which have a high frequency of introgression during domestication or polyploidization (Jobst et al. 1998). However, ITS intraspecies variation does not appear to be common in undomesticated, non-hybridized species (Ainouche and Bayer 1997). For those species of Echinacea that appear to result from interspecies hybridization (e.g. E. simulata), phylogenetic relationships may be inferred from the multiple sequence differences present in the ITS regions of the interspecies hybridization progenies when compared to the same regions of the parental species.
The sequence of Echinacea ITS2 shows fewer variable sites between species than ITS1. The ITS1 and ITS2 sequences may be useful in constructing phylogenetic trees that depict the taxonomic relationships between the organisms (FIG. 2). The alignment on which these trees is based is anchored by a highly conserved sequence, GGCRY-(N4-7)-(GY)2CAAGGAA, located in ITS1 (Liu and Schardl 1994). In FIG. 2, neighbor-joining dendrograms are based on distances computed with 2-parameter substitution rates, however unweighted parsimony trees produced similar topologies.
The number of publicly available ITS sequences of Echinacea is limited. More importantly, due to interspecies variations, it is unclear how well the published sequences of the Echinacea ITS regions represent Echinacea specimens obtained from the market. In order to develop a methodology that is suitable for identification and validation of Echinacea species in the market place, it is important to analyze sequences of the ITS regions using Echinacea specimens obtained from the market or from nature.
The samples to be tested (referred to as “target samples” in this disclosure) according to the disclosed methodology may range from fresh specimens to dried samples. These samples may be taken from roots, stems, leaves, flower or seeds of the plants. The disclosed methods are also suitable for verifying the identity of species contained in samples from previous growing seasons that have been kept in a warehouse. Typically, the samples are washed and pulverized before genomic DNA is extracted from the samples. Because the extraction process may cause damages to the integrity of the genomic DNA, DNA polymerases, such as the Klenow Fragment, may be used to repair such damages. The repairing process may fix gaps on the genomic DNA and may facilitate the subsequent PCR which uses the genomic DNA as a template.
The following examples illustrate the present invention. These examples are provided for purposes of illustration only and are not intended to be limiting. The chemicals and other ingredients are presented as typical components or reactants, and various modification may be derived in view of the foregoing disclosure within the scope of the invention. It is also recognized that although the examples below are given in the context of analyzing Echinacea samples, specimens from other plants may be analyzed with the disclosed methodology with or without modification. Nucleotide sequences disclosed herein may be presented as upper or lower case letters, both referring to the same nucleotide as is conventional in the art.
Example 1
Collection and Preparation of Samples for Nucleic Acid Analysis
More than one hundred specimens of Echinacea and Parthenium plants from across the growing range of these plants in the Midwest of the United States were collected. Table 1 is a list of specimens obtained through this endeavor. Some specimens were collected directly from the wild and kept as fresh samples, while others were dried samples that have been in storage. Genomic DNA was prepared from these specimens and DNA of the ITS regions was obtained by DNA amplification. More particularly, ITS1 primers were known to amplify the ITS genomic sequences from angiosperms and were synthesized according to Koopman et al., 1998 and Van der Stappen, et al., 1998. Each of ITS1 primer was separately modified by placing M13 and T7 sequences upstream of the sequence which is homologous to genomic DNA. Primers were also 5′ biotinylated to permit magnetic capture and purification of sequencing templates. This facilitated automated sequencing of both strands of the amplification product using commercially-available Cy5- and Cy5.5-labeled universal M13 and T7 primers.
Primer selection for amplification and/or sequencing was also facilitated by use of the methodology described in U.S. Pat. No. 5,848,492. We verified that the primer sequences reported by Van der Stappen et al. 1998 And Koopman et al. 1998 demonstrated high levels of average information content. Primers and amplification products were developed first by aligning and computing the average information for 28S ribosomal RNA gene sequences of flowering plant species. Oligonucleotide primers were selected, synthesized (by Oligos Etc, Wilsonville, Oreg.), and tested using methods described in Rogan et al. 1995 and Tooley et al. 1998. The amplified products were then sequenced using primers and methods as described in FIG. 1b of Van der Stappen et al. 1998 and primers prITS2 and prITS3. Primers prITS2 and prITS3 are complementary to each other and were used in separate sequencing reactions. Their sequences are shown below:








(SEQ ID NO. 82)











prITS2: 5′-gCT gCg TTC TTC ATC gAT gC-3′




 








(SEQ ID NO. 83)











prITS3: 5′-gCA TCg ATg AAg AAC gCA gC-3′







The resulted sequences were compared with one another by sequence alignment. Multiple sequences from the same genus were aligned to create a multialignment of the sequences.







TABLE 1







List of Specimens Collected








Sample ID*
Species





3001
E. pallida


3002
E. pallida


3003
E. pallida


3004
E. pallida


3005
E. pallida


3006
E. pallida


3007
E. pallida


3008
E. pallida


3009
E. pallida


3010
E. pallida


3011
E. pallida


3012
E. pallida


3013
E. pallida


3014
E. pallida


3015
E. pallida


3016
E. pallida


3017
E. pallida


3018
E. pallida


3019
E. pallida


3020
E. pallida


4021
E. atrorubens


4022
E. atrorubens


4023
E. atrorubens


4024
E. atrorubens


4025
E. atrorubens


4026
E. atrorubens


4027
E. atrorubens


4028
E. atrorubens


4029
E. atrorubens


4030
E. atrorubens


4031
E. atrorubens


4032
E. atrorubens


4033
E. atrorubens


4034
E. atrorubens


4035
E. atrorubens


4036
E. atrorubens


4037
E. atrorubens


4038
E. atrorubens


4039
E. atrorubens


4040
E. atrorubens


4041
E. atrorubens


4042
E. atrorubens


4043
E. atrorubens


4044
E. atrorubens


4045
E. atrorubens


4046
E. atrorubens


4047
E. atrorubens


4048
E. atrorubens


4049
E. atrorubens


4050
E. atrorubens


6051
P. integrifolium


6052
P. integrifolium


6053
P. integrifolium


6054
P. integrifolium


6055
P. integrifolium


3056
E. pallida


3057
E. pallida


3058
E. pallida


3059
E. pallida


3060
E. pallida


3061
E. pallida


2062
E. angustifolia


2063
E. angustifolia


2064
E. angustifolia


2065
E. angustifolia


2066
E. angustifolia


2067
E. angustifolia


2068
E. angustifolia


2069
E. angustifolia


2070
E. angustifolia


2071
E. angustifolia


2072
E. angustifolia


5073
E. paradoxa


5074
E. paradoxa


5075
E. paradoxa


5076
E. paradoxa


5077
E. paradoxa


5078
E. paradoxa


5079
E. paradoxa


5080
E. paradoxa


5081
E. paradoxa


5082
E. paradoxa


5083
E. paradoxa


5084
E. paradoxa


5085
E. paradoxa


5086
E. paradoxa


5087
E. paradoxa


5088
E. paradoxa


5089
E. paradoxa


5090
E. paradoxa


5091
E. paradoxa


5092
E. paradoxa


5093
E. paradoxa


5094
E. paradoxa


5095
E. paradoxa


5096
E. paradoxa


5097
E. paradoxa


5098
E. paradoxa


5099
E. paradoxa


2100
E. angustifolia


2101
E. angustifolia


2102
E. angustifolia


2103
E. angustifolia


2104
E. angustifolia


1105
E. purpurea


1106
E. purpurea


1107
E. purpurea


1108
E. purpurea


1109
E. purpurea


2110
E. angustifolia


2111
E. angustifolia


2112
E. angustifolia


2113
E. angustifolia


2114
E. angustifolia


2115
E. angustifolia


2116
E. angustifolia


2117
E. angustifolia


2118
E. angustifolia


2119
E. angustifolia





*A prefix of “PAT,” “PAT1_” or “1_” may be added before the 4-digit Sample ID in this Table without changing the identity of the sample.






FIG. 3 and FIG. 4 show the multiple alignment of sequences of the ITS1 region from Echinacea and Parthenium specimens, respectively. FIG. 5 shows the alignment of the ITS2 region from specimens from one single species, Echinacea angustifolia. Taken together, the data in FIGS. 3-5 and Table 1 indicate that while the DNA sequences of the ITS regions are well conserved among different species, there are significant interspecies variations among different species of Echinacea collected from nature. As demonstrated in FIG. 5, sequences of different organisms within the same species may also show some sequence variations.
Example 2
Design of PCR Primers for Specific Amplification of Either Echinacea or Parthenieum Species
The sequences of multiple Echinacea and Parthenium species were aligned (FIG. 6). After multiple alignment of all of the ITS1 and ITS2 sequences of all of the specimens, primers that were specific for either the Echinacea or Parthenium genus were selected based on inspection of the alignment. The primers were selected such that the primers would amplify most species of either the Echinacea or Parthenium genus, but not both. To this end, the 3′ termini of each primer was located at low information (non-conserved) positions, in which the sequence differences were maximized between the genera. Among the subspecies of Echinacea studied here, there were no differences in any of the sequences in the first 50 bp of the amplification product, or positions 100-150 bp. However there was sufficient divergence from the Parthenium sequence data to design PCR primers that may be used to distinguish the two genera.
Two sets of primers from each genus were selected based upon the methodology described above. The sequences of the resultant 4 sets of primers are as follows:








Primer set 1:









(SEQ ID NO. 84)









Forward: 5′- ACG GGG CGC AAT AGC ACG -3′



 








(SEQ ID NO. 85)









Reverse: 5′- CGA GGC CTT GTC GAC GTG TG -3′



 


Primer set 2:








(SEQ ID NO. 86)









Forward: 5′- TGT ATC CAT GAT GCC CCT AT -3′



 








(SEQ ID NO. 87)









Reverse: 5′- CAA GTA AAA CAC ATG ACC GAG -3′



 


Primer set 3:








(SEQ ID NO. 88)









Forward: 5′- GAC GGG GCA TAA CAG CAC AA -3′



 








(SEQ ID NO. 89)









Reverse: 5′- GTG AGG CCT TGT TGA CGA GC -3′



 


Primer set 4:








(SEQ ID NO. 90)









Forward: 5′- TGT TAG TGT GTC AAC CAG ACA -3′



 








(SEQ ID NO. 91)









Reverse: 5′- ACA TGT AAA ACT ACT GGC CTT T -3′







Example 3
PCR Assay for Identification of Echinacea Species
The 4 sets of primers described in Example 2 were tested for amplification at the manufacturer's recommended annealing temperature. Results indicated that the primers recognize their targets. However, under standard reaction conditions, the primers either did not amplify well, or showed a lack of specificity for all tested samples.
To determine the optimal reaction condition, a series of PCR reactions were set up under different annealing temperature and Mg2+ concentration. After optimization, the final conditions found to be best suited for amplification with primer sets 1 and set 4 were at 63 degrees for annealing and a master mix concentration of MgCl2 of 2.21 mM for set 4 and 1.91 mM MgCl2 for set 1. Each set of samples was amplified with two series of reactions per primer sets. One was expected to amplify Parthenium integrefolium series, the other was expected to amplify only species in the Echinacea series. Each series needed a positive control respective of its primers origin. Set 2 & 3 were also tested, and it was found that the highest possible annealing temperature which still routinely gives an appropriately sized product was about 64 degrees.
Tests were carried out on the primers once they had been properly diluted and aliquotted. The ITS1 primers (sets 1 and 4) that were ordered from IDT were for Echinacea sp. and Parthenium integrifolium sequences. Multiple species of Echinacea native to continental US were studied, and DNA from multiple exemplars were of each species was isolated. Multiple exemplars of P. integrifolium were also collected and DNA isolated. FIG. 7 is the result of such an experiment showing that the Echinacea specific primer sets 3 and 4 only generate PCR products when Echinacea DNA is present as template, while Parthenium specific primers, sets 1 and 2, only amplify Parthenium genomic DNA. Primers 3 and 4 may be used for Echinacea detection in a background of Parthenium or Rudbeckia contamination. Primers specific for either Echinacea or Parthineum may be employed to detect Echinacea or Parthineum in a mixture that may contain both species. The lane assignments and results shown in FIG. 7 are summarized in Table 2.







TABLE 2







Lane Assignments and PCR Test Results








Lane assignment:
Result:












1)
100 bp marker



2)
Parthenium-specific primers 100-200, sample 3015
−


3)
Parthenium-specific primers, sample 4027
−


4)
Parthenium-specific primers, sample 2066
−


5)
Parthenium-specific primers, sample 5077
−


6)
Parthenium-specific primers, sample 1106
−


7)
Parthenium-specific primers, sample 4023 (+)
−


8)
Parthenium-specific primers, sample (−control reaction,
−



no DNA)


9)
marker


10)
Echinacea-specific primers 100-200, sample 3015
−


11)
Echinacea-specific primers, sample 4027
+


12)
Echinacea-specific primers, sample 2066
+


13)
Echinacea-specific primers, sample 5077
+


14)
Echinacea-specific primers, sample 1106
+


15)
Echinacea-specific primers, sample 4024
+


16)
Echinacea-specific primers, sample (−control reaction,
−



no DNA)


17)
marker


18)
Parthenium-specific primers 100-200, sample 6052
+


19)
Parthenium-specific primers, sample 6053
+


20)
Parthenium-specific primers, sample 6054
+


21)
Parthenium-specific primers, sample 6055
+


22)
Parthenium-specific primers, sample 4023
−


23)
Parthenium-specific primers, sample (−control reaction,
−



no DNA)


24)
marker


25)
Echinacea-specific primers 100-200, sample 6052
−


26)
Echinacea-specific primers, sample 6053
−


27)
Echinacea-specific primers, sample 6054
−


28)
Echinacea-specific primers, sample 6055
−


29)
Echinacea-specific primers, sample 4023
+


30)
Echinacea-specific primers, sample (−control reaction,
−



no DNA)


31)
marker









FIG. 8 shows results of an experiment attempted to address the question of whether the source of DNA and the methods used in purifying the template DNA have any effects on the PCR amplification. Lanes 1 and 10 are DNA markers to ensure that the correct size product has been obtained. Lane 9 is a negative control reaction in which no DNA has been added; this ensures that the reagents have not been contaminated with any other DNA source. Lanes 2 (E. purpurea) and 3 (E. pallida) correspond to amplified DNAs isolated from fresh specimens collected during the year 2000 harvest (arrow). Lane 4 contains amplified DNA from a 5 year old Echinacea specimen (1996). Lanes 5 through 8 were amplification reactions of DNA from 2 different Echinacea species, each purified by both a commercial procedure (lanes 6 and 8) and another experimental procedure (lanes 5 and 7). No amplification was observed using the experimental DNA purification procedure, however amplification was detected with the commercial kit.
The results in FIG. 8 also show that DNA from powdered Echinacea purpurea derived from stored, aged preparations could be amplified with the Echinacea specific primer sets. A member of another closely related but distinct genus, Rudbeckia linti, was also tested with the Echinacea specific primers. No amplification product was produced with Echinacea specific primers and Rudbeckia DNA template.
Once the parameters for the primer sets had been finalized, the complete library of samples were tested to determine if there were any sub-species that were not compatible with the parameters, or if any artifactual amplification products might be produced. None were detected and all Echinacea DNA preparations were amplified. Because of the breadth of the geographic distribution of the samples and the fact that all relevant species were tested, we conclude that a “randomly sampled” plant taken from the field would produce the expected results with this assay primers.
We then carried out Tracer/Driver (T/D) experiments to determine the sensitivity of the assay to detect trace levels of contaminating Parthenium or Rudbeckia sequences in a presumed homogeneous preparation of Echinacea DNA. In these experiments, sample DNA needs to be diluted in known quantities in order to accurately determine the sensitivity of each primer set. The design uses primers to detect various levels of tracer DNA diluted into an excess of driver DNA. The Tracer DNA ranges in concentration from 10,000 to 1 genome equivalents (e.g.), so that each consecutive dilution will have a 10 fold lower concentration. Driver DNA will be added to qsp the weight of total DNA present per dilution to 50 ng for experimental reactions, while no Driver will be added to the Tracer positive dilutions. There will be two sets of T/D experimental dilutions per primer set. One set consists of Rudbeckia D DNA, and the other set consists of either Echinacea or Parthenium D DNA, depending on which primer set is being investigated. Primer Sets 1 & 4 were used. For primer set 4, the sensitivity of detecting Echinacea D DNA was 1000 fold lower when compared to the H2O ge dilutions. The sensitivity of detection with Set 1 was compromised only 10 fold when Parthenieum D was present and even less so when Rudbeckia D DNA was present. These results show a sensitivity of 16,000 g.e.'s for detection of Parthenium and 27,000 g.e.'s for detection of Echinacea. 
FIG. 9 shows results of agarose gel electrophoresis of amplified internal transcribed spacer sequences from several Echinacea species under different concentrations of Mg2+. DNA was extracted from washed, pulverized root material of Echinacea angustifolia (2 samples), E. atrorubens (2 samples), E. pallida (1 sample) and E. purpurea (3 samples). PCR amplification of the internal transcribed spacer ITS1 sequences of 23S rDNA using Primer Set 3 generated DNA products of appropriate size for all species except Echinacea pallida. The concentration of Mg2+ appears to have a drastic effect on the PCR results.
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## Claims

1. A pair of oligonucleotide molecules for amplification of ribosomal DNA from a plant material, said oligonucleotide molecules being selected from the group consisting of primer set 1 and primer set 2, wherein primer set 1 comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87.

2. A pair of oligonucleotide molecules for amplification of ribosomal DNA from a plant material, said oligonucleotide molecules being selected from the group consisting of primer set 3 and primer set 4, wherein primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

3. A method for determining the existence of an organism or its derivatives in a material, said method comprising:
(1) a first DNA amplification of a segment on a ribosomal DNA, wherein a first primers pair is used as PCR primer and DNA from at least one species belonging to the same genus as said organism is used as template;
(2) sequencing the PCR product resulting from the first amplification;
(3) a second DNA amplification using as PCR primers a second primer pair and using as template DNA prepared from said material;

wherein the selection of the first primer pair comprises the steps of:
(a) searching for a divergent segment of the DNA from said at least one species with low average information content determined quantitatively surrounded by two conserved segments of said DNA with high average information content determined quantitatively; and
(b) designing the first primer pair for PCR amplification of said divergent segment by constructing a sequence logo for said DNA such that said primers contain a set of sequences present in said sequence logo that encompass the nucleotide variability of said conserved segments, said primer pair being able to anneal to said conserved segments for amplification of said divergent segment;
and the selection of the second primer pair comprises the steps of:
(a) searching the sequences obtained from step (2) for at least one segment of DNA with interspecies variations; and
(b) designing the second primer pair for DNA amplification, wherein said second primer pair comprises at least one interspecies variation;
and

(4) determining the existence of said organism or its derivatives in said material.

4. The method of claim 3, wherein the PCR product resulting from the first amplification comprises an internal transcribed spacer (ITS) region.

5. The method of claim 3, wherein the second primer pair is selected from the group consisting of primer set 1, primer set 2, primer set 3, and primer set 4; and primer set 1 comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87, primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, and primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

6. A method for determining the existence of an Echinacea species or its derivatives in a sample based on primary structure of DNA from said Echinacea, said method comprising:
(1) amplification of a segment of the internal transcribed spacer region of the ribosomal DNA of said plant material, and
(2) determining the existence of said Echinacea species or its derivatives in said sample,
said amplification being performed using a pair of oligonucleotides selected from the group consisting of primer set 1, primer set 2, primer set 3, and primer set 4, wherein primer set 1 comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87, primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, and primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

7. The method of claim 6 wherein the amplified segment comprises at least one DNA fragment from an internal transcribed spacer region selected from the group consisting of ITS1 and ITS2.

8. The method of claim 6 wherein the segment is amplified using primers that specifically amplify DNA from Echinacea species but do not amplify DNA from Parthenium species.

9. The method of claim 6 wherein the segment is amplified using primers that specifically amplify DNA from Parthenium species but do not amplify DNA from Echinacea species.

10. The method of claim 6 further comprising a step of sequencing said amplified segment.

11. The method of claim 6 wherein DNA prepared from the sample is used as a template for said amplification.

12. The method of claim 11 wherein said sample has been stored for at least 6 months.

13. The method of claim 11 wherein the sample contains specimen from at least two species selected from the group consisting of Echinacea, Parthineum and Rudbeckia.

14. The method of claim 11 wherein the sample contains specimen from Echinacea and Parthineum.