JPH0349698A - Oligonucleotide for detecting bacteria and method for detecting bacteria using the same - Google Patents

Abstract

PURPOSE:To enable simple and rapid detection with a high sensitivity by carrying out polymerase chain reaction(PCR) treatment using a specific oligonucleotide functioning as a primer for nucleic acid synthetic reaction. CONSTITUTION:Microbial cells of the genus Salmonella are subjected to extraction treatment to provide a specimen (A) composed of a nucleic acid ingredient. The same sequence as the base sequence of ara C gene of the microorganism of the genus Salmonella is then chemically synthesized to afford a synthetic nucleotide (B) composed of a sequence group expressed by the formula. The ingredients (A) and (B) and a thermostable DNA polymerase are added to a buffer solution to afford a reaction solution (C). The resultant ingredient (C) is then thermally denatured at about 94 deg.C, subsequently annealed and further subjected to polymerization reaction. The PCR is performed in about 42 cycles to afford an amplified nucleotide fragment (D). The resultant ingredient (D) is then subjected to agarose electrophoresis, etc., to calculate the length of the nucleotide fragment and selectively detect the bacterium classified into the genus Salmonella in the specimen.

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention is applicable to clinical tests, especially food poisoning tests. 4. In food inspection, Salaonella bacteria (Salmo
Bacteria belonging to the genus nella) are detected. [Prior art and problems] When the test material is patient's vomit, feces, food, or swabbing material, Salmonel! Before identifying the bacterium as a bacterium, operations including enrichment culture and confirmation culture must be performed. The time required for each culture stage is 18 to 24 hours, and the total time required is approximately 2 days and 1 person, which is a long time. Confirmation culture costs include TSI agar, S
Inoculate into IM medium, VP-MR medium and lysine decarboxylation test medium and culture at 37°C overnight. Therefore, it is time consuming, expensive, and operationally complicated. On the other hand, recently, DNA probe methods or piperidization methods using oligonucleotides have been attempted. However, when detecting oligonucleotides by hybridization on a spread or other support using labeled-modified probes, it is difficult to obtain sufficient detection sensitivity and selectivity for bacterial introduction tests. [Purpose of the Invention] The present invention is directed to the production of Salsone by gene amplification technology using oligonucleotides as one primer in a nucleic acid synthesis reaction.
The purpose of this invention is to provide a simple, rapid, and highly sensitive method for testing Salsonella fields, which detects nucleic acids derived from +1a bacteria. [Means and effects for solving the problems] The present invention is characterized in that Salmonella bacteria is selectively detected by a gene amplification method in which an oligonucleotide functions as a primer. Regarding the gene amplification method, Polymera developed by Saiki et al.
chain reaction method (hereinafter abbreviated as PCR method; Science. 230.1) 50 (
(1985)). This method is based on a specific nucleotide sequence region (in the case of the present invention, Sal
When detecting the ara C gene of the B. nella bacterium, prepare oligonucleotides that recognize and hybridize the positive strand at one end of the region and the single strand at the other end, and denature the oligonucleotide by heat denaturation. Two mtl. 1 acid again
Separate into main strands and repeat the same reaction in 5. By repeating this series of operations, the copy number of the reliable region sandwiched between the two primers increases to the point where it can be detected. Specimens include clinical test materials such as feces, urine,
Blood, tissue homogenates, etc., and food materials may also be used. In order to use these materials as PCH samples, a pretreatment process is required to release nucleic acid components from the bacterial cells present in the materials. However, if there are several to tens of molecules or more of nucleic acids that can be piperidized by the primers, PCR will proceed, so the test material should be prepared using lytic enzymes, surfactants,
A sample solution with a sufficient amount of nucleic acid for PCR to proceed can be prepared by simply treating with alkali or the like for a short time. The oligonucleotide used as a primer in the present invention is a nucleic acid fragment having a length of 10 bases or more, preferably an istx group or more, in terms of selectivity, detection sensitivity, and reproducibility, and can be either chemically synthesized or natural. good. Furthermore, the primers do not need to be specifically labeled for detection purposes. Salsonel specified by 1 Reimer
The amplified region of the ara C gene of La bacteria ranges from 50 bases to 2,000 bases, preferably from 100 bases to 1.0 bases.
It should be 00 bases. Template-dependent nucleotide polymerization reaction uses n{thermal DNA polymerase,
The origin of this enzyme may be any biological species as long as it maintains its activity at a temperature of 90 to 95°C. The ripening temperature was 90 to 95°C, the temperature of the annealing operation for hybridizing the primer was 37 to 65°C, and the polymerization reaction was 50 to 75°C, and 20 to 42 cycles of the PCR reaction were performed, with this being one cycle. Amplify. For detection, the presence and length of the amplified nucleotide fragment can be confirmed by subjecting the enzyme reaction solution as is to agarose gel electrophoresis. From the results, it can be determined whether the target nucleotide is present in the sample. This determination directly determines the presence or absence of Salmonella bacteria. Electrophoresis and chromatography are also effective for detecting enriched nucleotide fragments. 7
Using 14 strains of bacteria, each was inoculated into an appropriate Masuda medium and cultured overnight at 37°C under aerobic conditions.
Bacterial cells were collected from 1.5 ml by centrifugation. 1
lr with 0mM Tris-HCl buffer (PH 7.5)
After washing with gJ, add 1 mg/m of lysozyme to the same protective solution.
Suspend in 0.5 ml of the solution dissolved to make 3
Bacteria were lysed at 7°C for 10 minutes. The same volume of phenol saturated with the Mfli solution was added to the lysate, and the mixture was stirred well. After centrifugation, collect the upper layer. Nucleic acid components are precipitated by ethanol precipitation treatment, and the precipitate is added to the above-mentioned solution, l
ml and used this as the sample. One mer ▲ Base sequence of Salmonella affl ara C gene (CI. xrke, P., et al.;
Gene 18, 157-163 (1982))
From the above, select the arrangement shown in claim 2,
An oligonucleotide with the same sequence was chemically synthesized. Chemical synthesis uses Shimadzu DNA synthesis 1fINS-1,
This was done using the triester method. Purification of the synthesized nucleotide fragments was performed using a Cla reverse phase column. Add 3 μl of the sample solution to 16.05 ml of sterile distilled water.
μ! , 10x reaction buffer - 3μ1, dNTP solution 4
．． 8μ1, primer (1) 1.5μ1, primer (
2}1, 5μ1 and ripening-resistant DNA polymerase 0.
Add 15μl, 30μl! A reaction solution was prepared.
Add mineral oil (SIGMA) to the container containing this reaction solution.
Add 50 µl of 50 μl of 100% of the solution (manufactured by Co., Ltd.) and layer it on top of the reaction solution. The contents of each added liquid are shown below. 10x reaction bag 7y: 500mM KC
I, 100mM Tris-HCI (pH 8.3
>, 1 5mM MgC I2, 0. 1% (
w/v) Gelatin dNTP solution: dATP. dCTP, dG
A mixture of TP and dTTP, each with a final concentration of 1.25 m
M Primers (1) and (2): Each water solution (500 (1/ml) of the chemically synthesized purified product described above) The combination of primers has the sequence (
From (a> to (c)), we used the following combination: Mar + Mar (a) + (b) (.L
〉+(c} Thermostable DNA polymerase: Taq DNA polymerase (5 units/ml; Perkin
(manufactured by Elaer Cetus) The reaction conditions are as follows. Thermal denaturation: 94°C 1 minute annealing = 37°C 1 minute Polymerization reaction: 60″Cl minute The process from thermal denaturation to polymerization reaction via anneal ring
cycle (required time 5.7 minutes), this is 42 cycles (total required time approximately 4 hours rff! )went. These operations were performed by Perkin Elmer Cetus Company'! A.D.
The reaction was carried out by programming the above reaction conditions into the NA Thermal Cycler. In order to detect the enriched nucleotide fragments from the stopper elution reaction solution, agarose electrophoresis was performed as follows. The agarose gel used had a gel concentration of 2% (w/v) and contained ethidium bromide (0.5 μg/ml). The electrical conditions for electrophoresis are a constant voltage of 100 V.
, &9 went for 30 minutes. The operating method and other conditions are described in Maniatis et al., Molecular Cloni
(1982). In addition to the reaction solution, a molecular weight marker was also run at the same time, and the length of the nucleotide fragment was calculated by comparing the relative mobility. Direction 1 As mentioned above, the nucleotide sequence of the ara C gene has already been determined, and the oligonucleotide of the present invention, that is, the primer, is used by PCR! ! The size of the nucleotide that causes lta can be estimated. according to it,
Primers (a) and (b) have 329 bases, (a
) and (c), nucleotides with a length of 539 bases should be amplified. The numerical values shown in Table 1 are the results of measuring the length of the nucleotides amplified by the above method, and the unit is kilobase pairs. As can be seen from the table, each combination of primers matches the estimated nucleotide length, indicating that they are correctly amplifying the targeted region of the C gene. Table 1 (a) + (b) (a) + (c) Old 22l: Ministry of Agriculture, Forestry and Fisheries 32 Livestock Hygiene Laboratory (Example 2) The results obtained in Example 1 are as follows: .SalIl+one
In order to confirm whether it is selective for B. lla bacteria, we conducted a comparative study of bacterial species other than Sal + wonella bacteria that could be tested in the B-bed test. The method is the same as that shown in Example 1, but (2)
．． (6). For the strain (1 g) under anaerobic conditions,
We have prepared samples that can be used in PCR by culturing at 40°C overnight. Bacteria cultured in specimen preparation are shown in Table 2.
These are the 11 strains shown in the vertical headings. In addition, human placenta-derived DNA was prepared at a concentration of 1 μg/mI.
IFJ was also subjected to PCR in the same manner. Table 2 shows the results.
It is shown in As in Table 1, the units of numbers in squares are kilobase pairs. Amplified nucleotide fragments, which appear to be by-products of PCH, were detected in some bacterial species, but all of them differed in length from the nucleotide fragments estimated from the FA base sequence of the ara C gene. Salaonel
If these bacterial species have the same araC gene as the Lyu seedlings, the nucleotides should have the same length as the results of Example 1. Therefore, it is clear that the amplified nucleotide fragments derived from these bacterial species were not produced by recognizing the ara C gene, and Salmonell
It can be seen that it can be easily distinguished from and detected from A bacterium. In addition, if the agarose electrophoresis used in the examples of the present invention is performed under the above-mentioned electrophoresis conditions, if the range is 100 base pairs or less, it will be 5 to 104 base pairs, and if the range is 100 to 500 base pairs, it will be 10 to 104 base pairs. Can distinguish between 20 base pair nucleotide length differences. Furthermore, if the accuracy of nucleotide length measurement is improved by using a gel such as acrylamide, it is thought that the reliability of selective detection will further increase. (Leaving space below) Table 2 Combination of strain name Brimer (a) + (b) (a) + (c)! ;almonella typhig+urium (IFO12529) 0.33 0.54 Bacillus cereus Ca+apylobacter jeJuniEsc
herichia coli Vibr[o parihaemolyticusS
tapylococcus aureus Clost
ridius perfringensShigeordera dysenteriaeShigella fl
exnerlShigella sonnei (1)0.20 (2) (3)0.50 (4)0.50 (5)0.10 <6> (7)0.80 (8)0.50 (9)0.80 0.20 0.40 0,20 0.20 Human placenta (12) Gollectlxn, (12 冫Humanpface
ntal DNA Manufactured by Onco [Effects of the Invention] In the present invention, by using the PCR method, S
When detecting B. ella , it is possible to obtain high detection sensitivity due to the gene amplification effect and high selectivity due to the reaction being defined by two or more primers. In addition, because of its high detection sensitivity, it does not require a large amount of specimen, and pretreatment of the specimen is simple. Furthermore, the reaction time is short, detection requires only simple equipment, and operation is easy, so the time required for identification can be significantly shortened. As shown in the following example, the reaction time was 4 hours and the detection operation was 30 minutes. Also. For detection, agarose gel electrophoresis and nucleic acid staining with ethidium are used. Detection can be performed without labeling primers, etc. Furthermore, since the length of the nucleic acid can be precisely 2"2, the results are highly reliable. It is thought that the ara C gene of Salmonella bacterium is present ubiquitously in all idle strains belonging to the order Sala+one. Therefore, by targeting this gene, S^lsone l Iaf
fl G FJ can be detected all at once. On the other hand, since the nucleotide sequence has little homology with the ira C gene of other species, selectivity against Salmonella bacteria is maintained.

Claims (7)

[Claims] (1) Oligonucleotides for selectively detecting bacteria belonging to the genus Salmonella in a sample, or oligonucleotides that target the nucleotide sequence encoding the araC gene of Salmonella bacteria, and chemically A synthesized oligonucleotide, wherein the synthetic nucleotide has the following sequence group, (5') d-GGCGAGCAGTTTGTCTGTC
(3')...(a) (5')d-TACCGCCATACGTCTGAGC
(3')...(b) (5')d-GTTTCGCCTGGCTGATACG
(3')...(c) or a corresponding complementary sequence. (2) An oligonucleotide comprising at least 10 or more consecutive bases among the sequences of each oligonucleotide described in claim 1. (3) A method characterized in that an oligonucleotide having one of the oligonucleotide sequences set forth in claim 1 functions as a primer for a chain length reaction to selectively amplify a target nucleotide sequence. (a) A primer is hybridized to the single-stranded target nucleotide sequence in the sample to perform a chain length reaction by polymerization reaction of four types of nucleotides, (b) the obtained double-stranded nucleotide sequence is When separated into single strands, the complementary strand functions as a template for the chain length reaction by the other primer, (c) simultaneous chain length reaction by these two primers,
By repeating separation of the long-chain product from the template and hybridization with new primers, a specific nucleotide sequence is amplified, which is detected by electrophoresis and chromatography; (d) As a result, the aforementioned specimen Salmonell
A method comprising detecting bacteria. (4) The presence of the sequence to be recognized in the sample is determined by separating the nucleotide sequence amplified by electrophoresis or chromatography from the reaction product in the method according to claim 3 and determining its molecular weight. A method comprising detecting Salmonella bacteria by determining whether Salmonella bacteria are present. (5) A detection method by performing agarose electrophoresis and nucleic acid staining with ethidium bromide from the reaction product in the method according to claim 3. (6) A method for selectively detecting a target nucleotide sequence on a membrane or other support by using an oligonucleotide having one of the oligonucleotide sequences described in claim 1 as a probe. (7) The method according to claim 6, wherein the oligonucleotide having one of the oligonucleotide sequences described in claim 1 is modified with a label.