Method for testing causative microorganism of food poisoning and reagents therefor

Abstract: A method for testing causative bacterial species of food poisoning which is characterized by using two oligonucleotide primers that hybridize to opposite strands of bacterial DNA specifically, and flank a unique region in the target DNA and amplifying the specific fragment of the bacterial DNA, comprising the steps of: (a) hybridizing the primer to specific gene sequence of bacteria in a sample, extending the hybridized primer with deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and resultantly making the double strand nucleotide; (b) where the primer extension products are cleaved into each single strand of nucleotide by certain external force such as heat, pH and so on, one single strand functioning as a template for nucleotide extension with a primer of the other strand; (c) repeating a series of cycles involving cleavage of primer extension products, primer hybridizing, extension of the hybridized primers to amplify the specific fragment of DNA, and detecting the amplified DNA fragment; and (d) as the result, determining whether or not the specific fragment is present in said sample, thereby to confirm a species of causative bacteria of food poisoning.

Description: FIELD OF THE INVENTION The present invention relates to detection of a species of causative bacteria of food poisoning in clinical tests or food tests. STATEMENT OF THE PRIOR ART Where materials to be tested are patients' excreta or feces, food or wiping materials, operations of enrichment culture, separation culture, pure culture and then confirmation culture must be carried out, in order to identify that certain bacteria are causative bacteria of food poisoning. Since a time period required for each culture step is 18 to 24 hours, the required period becomes as long as about 4 days in total. In biochemical tests for confirmation culture, for example, egg-yolk reaction, VP reaction, gelatin liquefaction, starch hydrolysis, nitrate reduction and sugar reduction, etc. must be examined. Therefore, the biochemical tests are operationally complicated. Accompanied by the complicated operations, the biochemical tests are time consuming and expensive. On the other hand, DNA probing technique or hybridization technique using oligonucleotide has been attempted in recent years ( cf. JOURNAL CLINICAL MICROBIOLOGY vol. 27, no. 2, February 1989, pages 261 - 265 OLIVE,M. 'Detection of Enterotoxigenic Escherichia coli after polymerase chain reaction' and JOURNAL OF BACTERIOLOGY vol. 171, no. 3, March 1989, pages 1602 - 1608 SHYAMALA, V ET AL. 'Amplification of bacterial genomic DNA by polymerase chain reaction', ). However, there are difficulties to achieve satisfactory detection sensitivity and selectivity in bacterial tests. SUMMARY OF THE INVENTION The present invention relates to detection of nucleic acid derived from Bacillus cereus , a bacteria causative of food poisoning, by gene amplification technique in which oligonucleotide functions as a primer for synthetic reaction of nucleic acid. An object of the present invention is to provide a method and a reagent for testing for B . cereus , a microorganism causative of food poisoning, in a simple and rapid way with high sensitivity. This object is achieved by a method as set forth in claim 1 and a reagent as defined in claim 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Herein, the gene amplification of the present invention is performed based on the polymerase chain reaction (hereafter simply referred to as PCR method; Science, 230, 1350 (1985)) developed by Saiki et al. According to this method, where a specific nucleotide sequence region is to be detected, such oligonucleotides that one of the both ends in the region recognizes + chain and another recognizes - chain to effect hybridization are prepared. The oligonucleotides are allowed to function as primers for template-dependent nucleotide polymerization of a nucleic acid test sample which is changed to a single stranded state by thermal denaturation. The resulting double stranded nucleic acid is again separated into a single strand and a similar reaction is again caused. By repeating a series of operations, the region inserted between the two primers is so amplified as to have the number of copies, whereby the region is detectable. The nucleotide sequence region of primers varies depending upon species of causative microorganisms of food poisoning. The following sequence regions correspond to Bacillus cereus: As a test sample, materials for clinical tests such as feces, urine, blood, tissue homogenate or the like and food materials may be used. To use these materials as test samples for gene amplification, an operation for releasing the nucleic acid component from bacteria present in the materials is required as a pretreatment. However, since more than several ten molecules of nucleic acid hybridizable with the primer are present, the gene amplification process, a sample solution having nucleic acid in an amount sufficient to proceed the gene amplification can be prepared merely by treating a test material with bacteriolysin, a surfactant, an alkali, etc. for a short period of time. The oligonucleotides having the sequences described above which are used as primers in the present invention are nucleic acid fragments having a size of more than 10 nucleotides, preferably more than 15 nucleotides, in view of their selectivity, detection sensitivity and reproducibility. The oligonucleotides may be either chemically synthesized or natural ones. The primers are not necessarily labeled especially for detection. The amplified sequence in a specific species of causative bacteria of food poisoning governed by the primer may be between 50 and 2000 nucleotides, desirably between 100 and 1000 nucleotides. For the template-dependent nucleotide polymerization, thermostable DNA polymerase is used. This enzyme may be derived from organism of any source, so long as the enzyme can maintain its activity at a temperature of from 90 to 95°C. A temperature for thermal denaturation is between 90 and 95°C; a temperature for annealing to hybridize the primer is between 37 and 65°C; and a temperature for the polymerization is between 50 and 75°C. These procedures are made one cycle for gene amplification and, gene amplification is carried out in 20 to 42 cycles to effect amplification. Detection is made by applying the enzyme reaction solution to agarose gel electrophoresis as it is, whereby the presence of nucleic acid fragment amplified and its size can be confirmed. From the results, it can be judged if nucleic acid having the sequence to be recognized by the primer is present in a test sample. This judgment also applies to the presence or absence of causative bacteria of food poisoning as it is. For detection of the amplified nucleic acid fragment, other electrophoresis or chromatography is also effective. The oligonucleotides having the sequences (a) through (e) described above can selectively detect specific gene sequence of Bacillus cereus as a causative bacteria of food poisoning and hence can be used as reagents for tests. [Detection of Bacillus cereus] (Example 1) Preparation of test sample Using 6 strains shown on the first row in Table 1 as Bacillus cereus, each strain was inoculated on an appropriate enrichment medium followed by culturing at 37°C overnight under aerobic conditions. From 1.5 ml of the medium, cells were recovered by centrifugal operation. After washing once with 10 mM Tris-hydrochloride buffer (pH 7.5), the cells were suspended in 0.5 ml of a solution of 1 mg/ml of lysozyme in the buffer to cause bacteriolysis at 37°C for 10 minutes. The same volume of phenol saturated with the above buffer was added to the bacteriolysis solution and the mixture was thoroughly agitated. After centrifugation, the supernatant was recovered and treated with ethanol to precipitate the nucleic acid component. The precipitate was dissolved in 1 ml of the above buffer and the resulting solution was used as a test sample. Synthesis of primer From the nucleotide sequences of β-lactamase I and II type genes for Bacillus cereus (Wang, W., et al.; J. Bacteriol., 163, 487-492 (1985); Hussain, M., et al.; J. Bacteriol., 164, 223-229 (1985)), the sequences shown in claim 1 and 3 were selected ((a), (b) and (c): from β-lactamase I type gene; (d) and (e): from β-lactamase II type gene) and oligonucleotides having the same sequences were chemically synthesized. The chemical synthesis was carried out according to the triester method, using DNA synthesizer, NS-1, manufactured by Shimadzu Corporation. The synthesized oligonucleotide fragment was purified using C18 reverse phase column. Gene amplification Using 3 µl of the sample solution described above, 16.05 µl of sterile distilled water, 3 µl of buffer for 10-fold reaction, 4.8 µl of dNTP solution, 1.5 µl of primer (1), 1.5 µl of primer (2) and 0.15 µl of thermostable DNA polymerase were added to the sample solution to prepare 30 µl of the reaction solution. To a container charged with the reaction solution, 50 µl of mineral oil (manufactured by SIGMA Co.) was added to form a layer on the reaction solution. The content of each liquid added is shown below. Buffer for 10-fold reaction: 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.1% (w/v) gelatin dNTP solution: a mixture of dATP, dCTP, dGTP and dTTP in the final concentration of 1.25 mM each Primers (1) and (2): an aqueous solution of each of the chemically synthesized and purified products (5 ODU/ml) As the primers, the following combinations were chosen from the sequences ((a) through (e)) shown in claim 1 and 3 and used. Primer (1) + Primer (2) (a) + (b) (a) + (c) (d) + (e) Thermostable DNA polymerase: Taq DNA polymerase (5 units/ml; manufactured by Perkin Elmer Cetus Co.) The reaction conditions are as follows: Thermal denaturation: 94°C, 1 minute Annealing: 37°C, 1 minute Polymerization: 60°C, 1 minute The procedure from the thermal denaturation to the polymerization via annealing was made one cycle (time required: 5.7 minutes) and 42 cycles of the procedure were repeated (total time required: about 4 hours). These operations were carried out by programming the reaction conditions described above on DNA Thermal Cycler manufactured by Perkin Elmer Cetus Co., Ltd. Detection In order to detect the amplified nucleotide fragment from the reaction solution, agarose gel electrophoresis was performed as follows. Agarose gel was used in a gel concentration of 2% (w/v). Agarose gel used contained ethidium bromide (0.5 µg/ml). The electrophoresis was carried out under electric conditions of 100 V in constant voltage for 30 minutes. The procedures and other conditions were the same as those described in Maniatis et al.; Molecular Cloning (1982). In addition to electrophoresis of the reaction solution, a molecular weight marker was also subjected to electrophoresis at the same time. By comparison in relative mobility, the size of nucleotide fragment in the gel detected by ultraviolet rays, etc. was calculated. Results As described above, the nucleotide sequences of both types I and II of β-lactamase gene were already determined. Therefore, the size of nucleotides amplified by the oligonucleotides of the present invention, namely, the primers, through gene amplification can be deduced. According to the deduction, the sizes of 156 nucleotides, 313 nucleotides and 232 nucleotides are to be amplified in primers (a) and (b), (a) and (c) and (d) and (e), respectively. The numerical values shown in Table 1 show the results obtained by measuring the size of nucleotides amplified by the method described above, wherein unit is kilo base pair. As is understood from the table, the respective combinations of the primers show numerical values identical with the deduced sizes of nucleotides, indicating that the region targeted by β-lactamase gene is correctly amplified. Combination of Primer Strain (a) + (b) (a) + (c) (d) + (e) Bacillus cereus 0.15 0.31 0.23 Bacillus cereus 0.15 0.31 0.23 Bacillus cereus 0.15 0.31 0.23 Bacillus cereus 0.15 0.31 0.23 Bacillus cereus 0.15 0.31 0.23 Bacillus cereus 0.15 0.31 0.23 Strain numbers (1) through (6) of Bacillus cereus and organizations from which the strains were acquired are shown below. (Example 2) In order to confirm as to if the results obtained in Example 1 is selective to Bacillus cereus, the results were compared and studied with other species than Bacillus cereus that could be tested in clinical tests. The same method as shown in Example 1 was used but with respect to strains (3) and (7), incubation was performed at 37°C overnight under anaerobic conditions to prepare test samples applicable to the gene amplification method. The strains cultured to prepare test samples are 7 species shown on the first row of Table 2. Human placental DNA was prepared in a concentration of 1 µg/ml and was also subjected to the gene amplification in a similar manner. The results are shown in Table 2. As in Table 1, unit of numerical values in the column is kilo base pair. In a part of the bacterial species, the amplified nucleotide fragments considered to be the by-products in gene amplification were detected but any of them has the size different from that of the nucleotide fragment deduced from the nucleotide sequence of β-lactamase gene. If these bacteria have the same β-lactamase gene as that of Bacillus cereus, the nucleotide fragment having the same size as the results of Example 1 must be detected. Accordingly, it is evident that the amplified nucleotides derived from these bacteria are not formed by recognizing β-lactamase gene and can thus be readily distinguished over and detected distinctly from Bacillus cereus. By performing agarose gel electrophoresis applied to the examples of the present invention under the aforesaid conditions, differences in size of nucleotide having 5 to 10 base pairs can be distinguished within the range below 100 base pairs and differences in size of 5 nucleotide having 10 to 20 base pairs can be distinguished within the range of from 100 to 500 base pairs. Furthermore, by increasing the accuracy of measurement in size of nucleotide using acrylamide, etc. as gel, reliability in the selective detection is considered to be more enhanced. Combination of Primer Strain (a) + (b) (a) + (c) (d) + (e) Bacillus cereus (JCM 2152) 0.15 0.31 0.23 Bacillus subtilis - - - Salmonella typhimurium 0.18 - 0.18 Campylobacter jejuni 0.20 - - Escherichia coli - - 0.18 Staphylococcus aureus - 1.00 0.40 Vibrio parahaemolyticus 0.33 - 0.70 Clostridium perfringens - - - Human placenta 0.28 - - Strain numbers (1) through (8) and organizations from which the strains were acquired are shown below. (Example 3) It is shown below up to what trace amount Bacillus cereus in the test sample could be detected according to the method of the present invention. Method Bacillus cereus (JCM 2152) was inoculated on 100 ml of enrichment medium followed by culturing at 37°C overnight under aerobic conditions. From the medium, cells were recovered by centrifugal operation. After washing once with 10 mM Tris-hydrochloride buffer (pH 7.5), the cells were suspended in 20 ml of lysozyme solution (1 mg/ml) in the buffer to cause bacteriolysis at 37°C for 10 minutes. To the lyzed solution was added 1 ml of 20% (w/v) SDS aqueous solution and the mixture was heated at the same temperature for 10 minutes. Then proteinase K was added thereto in a final concentration of 750 µg/ml followed by heating for further 30 minutes. The same volume of phenol saturated (with the above buffer) was added to the reaction mixture. After thoroughly agitating, the supernatant was recovered by centrifugation. Swine pancrease ribonuclease was added to the solution in a final concentration of 50 µg/ml followed by heating at 37°C for 60 minutes. The same volume of phenol described above was added to the solution. After thoroughly agitating, the supernatant was recovered by centrifugation. For ethanol precipitation, 3 M sodium acetate buffer (pH 5.2) in a volume of 1/10 that of the recovered solution was added to and mixed with the supernatant. Then ethanol was added to the mixture in a volume of twice that of the recovered solution followed by agitation. The precipitated thread-like DNA was recovered by centrifugal operation and again dissolved in 10 ml of 10 mM Tris-HCl (pH 7.5) buffer to obtain pure DNA standard. An amount of DNA in the standard was calculated based on its absorbance at a wavelength of 260 nm. One microgram of chromosomal DNA of Bacillus cereus corresponds to 3 x 10 8 Bacillus cereus. Thus, if how many numbers of Bacillus cereus are present, it can be detected by the amount of DNA incorporated into the gene amplification system. Gene amplification and other procedures are as shown in Example 1. As the combination of primers, (a) + (c) was used. Detection of the amplified nucleotide fragment was made by taking a picture of the electrophoresed gel on a transilluminator and confirming the band on a film. As a camera, Mamiya RB67 was used and a film was Polaroid type 667. Conditions for taking the picture: focus, 5.6; shutter speed, 1 second; Kenko RI filter was used. Results The results are shown in the picture below. Lane No. Amount of DNA (g) Number of Molecule (1) 10 -3 3 x 10 5 (2) 10 -4 3 x 10 4 (3) 10 -5 3 x 10 3 (4) 10 -6 3 x 10 2 (5) 10 -7 3 x 10 1 The desired band was confirmed also in Lane No. (5). This indicates that an amount of DNA corresponding to 30 molecules of chromosomal DNA of Bacillus cereus was detected. Theoretically, 30 pieces of Bacillus cereus are sufficient for the detection. According to the present invention, high detection sensitivity by gene amplification and high sensitivity due to the reaction controlled by two or more primers can be obtained in detection for causative microorganisms of food poisoning. Because of high detection sensitivity, large quantities of test samples are not required and pretreatment of test samples can be simplified. In addition, a reaction time is shortened and detection can be made merely by simple equipments. The operation is easy so that a time period required for identification can be greatly shortened. As demonstrated in the foregoing examples, it takes 4 hours for the reaction and 30 minutes for the operations for detection. Furthermore, by using agarose gel electrophoresis and nucleic acid staining with ethidium bromide, detection can be made without labeling the primers, etc. and the size of nucleic acid can be confirmed. Thus, reliability of the results is high. SEQUENCE LISTING SEQ ID NO:1 SEQUENCE LENGTH: 19 bases SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA HYPOTHETICAL: No ANTI-SENSE: No ORIGINAL SOURCE: ORGANISM: Bacillus cereus FEATURE: IDENTIFICATION METHOD: S SEQ ID NO:2 SEQUENCE LENGTH: 20 bases SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA HYPOTHETICAL: No ANTI-SENSE: No ORIGINAL SOURCE: ORGANISM: Bacillus cereus FEATURE: IDENTIFICATION METHOD: S SEQ ID NO:3 SEQUENCE LENGTH: 18 bases SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA HYPOTHETICAL: No ANTI-SENSE: No ORIGINAL SOURCE: ORGANISM: Bacillus cereus FEATURE: IDENTIFICATION METHOD: S SEQ ID NO:4 SEQUENCE LENGTH: 20 bases SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA HYPOTHETICAL: No ANTI-SENSE: No ORIGINAL SOURCE: ORGANISM: Bacillus cereus FEATURE: IDENTIFICATION METHOD: S SEQ ID NO:5 SEQUENCE LENGTH: 20 bases SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA HYPOTHETICAL: No ANTI-SENSE: No ORIGINAL SOURCE: ORGANISM: Bacillus cereus FEATURE: IDENTIFICATION METHOD: S

Claims: A method for testing causative bacterial species of food poisoning by polymerase chain reaction (PCR) technology by using two oligonucleotide primers that hybridize to opposite strands of a specific gene sequence of bacterial DNA specifically, and flank a unique region in the target DNA and amplifying the specific fragment of the bacterial DNA, characterized in that said bacterial species of food poisoning is Bacillus cereus and said specific gene of the bacteria is β-lactamase gene of Bacillus cereus, wherein each synthetic oligonucleotide defined below (a)-(e) is used as a primer of extension, and its nucleotide sequence is complementary to a sequence of the β-lactamase gene : wherein (a) is used together with (b) or (c) and (d) is used together with (e). A method as defined in claim 1 wherein detection of said specific fragment is carried out by agarose gel electrophoresis and staining of nucleic acid with ethidium bromide. A reagent for testing for Bacillus cereus, which reagent comprises two synthetic oligonucleotides, targeting the β-lactamase gene of Bacillus cereus, and being complementary to a sequence of said β-lactamase gene, said synthetic oligonucleotides being selected from the following group of sequences: wherein (a) is to be used together with (b) or (c) and (d) is to be used together with (e).

Inventors: ABE HIROHISA, AOYAMA YOSHIHIRO, FUKUSHIMA SHIGERU, JIKUYA HIROYUKI, OHASHI TETSUO, SHIRASAKI YOSHINARI, TADA JUN, TAKANO JUN, YAMAGATA KOICHI, ABE, HIROHISA, AOYAMA, YOSHIHIRO, FUKUSHIMA, SHIGERU, JIKUYA, HIROYUKI, OHASHI, TETSUO, SHIRASAKI, YOSHINARI, TADA, JUN, TAKANO, JUN, YAMAGATA, KOICHI
Applicants: SHIMADZU CORP, SHIMADZU CORPORATION
Application year: 1990
Main classification: C12Q1
IPC codes: C12Q1