CN102816842A - Multiplex PCR-DHPLC detection primers, kit and detection method for Vibrio parahemolyticus - Google Patents
Multiplex PCR-DHPLC detection primers, kit and detection method for Vibrio parahemolyticus Download PDFInfo
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Abstract
The invention discloses multiplex PCR-DHPLC detection primers, a multiplex PCR-DHPLC kit and a multiplex PCR-DHPLC detection method for Vibrio parahemolyticus. The detection primers are represented by SEQ ID No. 1, 2, 3, 4, 5 and 6. With the MPCR-DHPLC detection method provided by the invention, detection for specificity and a virulence gene of Vibrio parahemolyticus in a sample to be detected can be finished in one amplification reaction, so whether the sample to be detected contains Vibrio parahemolyticus or not and whether the Vibrio parahemolyticus contains a virulence gene or not are conveniently, rapidly and accurately determined. When MPCR-DHPLC detection is carried out on a sample by using the detection primers and the detection kit according to the detection method provided by the invention, the advantages of rapid and reliable detection, high sensitivity and strong specificity can be obtained. Therefore, the detection primers, the detection kit and the detection method provided by the invention are applicable to inspection and quarantine of food, seafood and the like, especially of import and export food and seafood, and can be used by an inspection and quarantine bureau, a center for disease control and prevention and a quality supervise department for convenient, rapid and accurate detection of samples.
Description
Technical field:
The invention belongs to technical field of bioengineering; Multiplex PCR-the DHPLC that is specifically related to important foodborne bacterial pathogens Vibrio parahemolyticus (Vibrio Parahemolyticus) detects primer, test kit and detection method, is suitable for import and export food check, CDC, quality supervised department use.
Background technology:
Vibrio parahaemolyticus (Vibrio Parahaemolyticus) is that a kind of Gram-negative is had a liking for the salt bacillus, is one of important foodstuffs poisoning induced germ of maritime nation, can cause patient's diarrhoea, enterospasm, feels sick, typical gastro-enteritis reactions such as vomiting, fever.Vibrio parahemolyticus mainly is present in seawater and the sea-food, accounts for 40% ~ 60% of bacterial food poisoning in Japan by this microbial food poisoning, occupies the first place.In addition, the U.S., Australia, Britain, Belgium, France, Korea S and south east asia all have this bacterium to cause the report of food poisoning outburst.The data of China since 1998 shows that generation scale and crowd's exposure scale of the food poisoning that Vibrio parahaemolyticus causes are obvious ascendant trend, all surpassed salmonella food poisoning, the first place of leaping to bacterial poisoning.
The Vibrio parahemolyticus traditional detection method is based on morphological specificity, dyeing characteristic, biochemical character, serotype isophenous characteristic and identifies.These conventional methods have played vital role in the Vibrio parahemolyticus research work; But development of modern science and technology; Traditional dependence phenotypic characteristic is identified the method for Vibrio parahemolyticus, can not satisfy far away the quick diagnosis of this bacterium and the needs of epidemiological study, exposes many weak points: complex operation; Qualification cycle is long, is unfavorable for the quick diagnosis of acute poisoning and the requirement that the port speeds passage through customs; The pathogenesis of Vibrio parahemolyticus is complicated and diversified; The bacterial strain that does not contain the virulence factor not necessarily causes a disease; And the virulence that can't confirm this bacterium in traditional culture identification method short period of time is strong and weak, also need continue to increase other special identification experiments such as biochemistry, and this makes qualification cycle veryer long undoubtedly.Therefore, urgent be badly in need of working out can be quicker, accurate, detect that flux is higher, artifical influence factor reduces as far as possible, and can obtain multiple detection of information methods such as isolating pathogen gene type, virulence gene distribution.
Dhplc analysis (Denaturing High-Performance Liquid Chromatography; DHPLC) be the new technology of setting up in recent years and being able to develop rapidly, be widely used in a plurality of fields such as SNP and mutation analysis, methylation analysis, the analysis of dna fragmentation length.
Summary of the invention:
The purpose of this invention is to provide a kind of Vibrio parahemolyticus multiplex PCR-D HPLC quick, reliable, highly sensitive, high specificity and detect primer, test kit and detection method, specific detection and the virulence gene that just can accomplish Vibrio parahemolyticus through primary first-order equation detect.
The present invention is a goal gene with toxR, tdh, the trh of Vibrio parahemolyticus (VP); Detect its tdh, trh virulence gene simultaneously with MPCR-DHPLC specific detection VP; Thereby differentiate Vibrio parahemolyticus (VP) and virulence gene thereof, realized the object of the invention.
Vibrio parahemolyticus multiplex PCR of the present invention-DHPLC detects primer, it is characterized in that, described detection primer is as follows:
To the tdh gene: tdh F5 '-gcaccggtcaatgtagaggt-3 ', (shown in SEQ ID NO.1)
Tdh R5 '-cagcagaatgaccgtgctta-3 '; (shown in SEQ ID NO.2)
To the trh gene: trh F5 '-aactgaatccccggttaagg-3 ', (shown in SEQ ID NO.3)
Trh R5 '-atatgtccattgccgctctc-3 '; (shown in SEQ ID NO.4)
To the toxR gene: toxR F5'-tgatttgcgggtgatttaca-3', (shown in SEQ ID NO.5)
toxR?R5'-ccgtctttagcgacgacttc-3'。(shown in SEQ ID NO.6)
Vibrio parahemolyticus multiplex PCR of the present invention-DHPLC detection kit comprises the MPCR reaction solution, detects primer and DHPLC reaction reagent, it is characterized in that,
Described detection primer is as follows:
To the tdh gene: tdh F5 '-gcaccggtcaatgtagaggt-3 ', (shown in SEQ ID NO.1)
Tdh R5 '-cagcagaatgaccgtgctta-3 '; (shown in SEQ ID NO.2)
To the trh gene: trh F5 '-aactgaatccccggttaagg-3 ', (shown in SEQ ID NO.3)
Trh R5 '-atatgtccattgccgctctc-3 '; (shown in SEQ ID NO.4)
To the toxR gene: toxR F5'-tgatttgcgggtgatttaca-3', (shown in SEQ ID NO.5)
toxR?R5'-ccgtctttagcgacgacttc-3'。(shown in SEQ ID NO.6)
Vibrio parahemolyticus multiplex PCR-DHPLC the detection method of non-diagnostic purpose of the present invention is characterized in that, may further comprise the steps:
(1) sample is increased bacterium and cultivate, the genomic dna that extracts thalline in the enrichment liquid is as template;
(2) use above-mentioned detection primer to tdh gene, trh gene and toxR gene; Mix the formation amplification reaction system with the MPCR reaction solution; Carry out the MPCR reaction; After reaction finishes, the PCR reaction product is carried out DHPLC analyzes, according to the DHPLC analytical results judge whether to contain Vibrio parahemolyticus with and the virulence gene somatotype.
Described amplification reaction system is preferably the system of 50 μ l, comprises 10 * Buffer6 μ l, dNTPs5 μ l; To each 1 μ l of upstream and downstream primer of tdh and toxR gene, to each 3 μ l of upstream and downstream primer of trh gene, Taq enzyme 1U/ μ l0.5 μ l; Distilled water 26.5 μ l, template DNA 2 μ l.
Describedly carry out MPCR reaction, be preferably, its loop parameter is that 95 ℃ of 30s, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations, and 72 ℃ are extended 10min.
It specifically is to get 5 μ L PCR products to carry out the DHPLC analysis that the described DHPLC of carrying out analyzes; Select non-sex change-double-stranded DNA multi-disc section clastotype, 50 ℃ of column temperatures, moving phase is that buffered soln A volume(tric)fraction is 50.2%; Buffered soln B volume(tric)fraction is 49.8%, flow velocity 0.9ml/min; Described buffered soln A is that the 0.1mol/L TEAA aqueous solution, buffered soln B are the acetonitrile solution that 0.1mol/LTEAA contains volume(tric)fraction 25%.
The present invention is with the target gene of toxR gene as the Vibrio parahemolyticus specific detection; With tdh gene and trh gene as whether pathogenic virulence detects gene; Design detects primer; Detect according to MPCR-DHPLC detection method of the present invention; In amplified reaction, accomplish specific detection and virulence gene detection to the Vibrio parahemolyticus of testing sample, thus easy, treat and whether contain Vibrio parahemolyticus and this Vibrio parahemolyticus in the sample article and whether contain virulence gene and judge fast and accurately.With detection primer of the present invention and detection kit, according to detection method of the present invention sample is carried out MPCR-DHPLC and detect, detect quick, reliable, highly sensitive, high specificity.Therefore the present invention is applicable to inspection and quarantine such as food and sea-food, especially to import and export food and sea-food, can supply inspection and quarantine bureau, disease prevention and control center and quality supervised department that sample is carried out easy, accurate detection.
Description of drawings:
Fig. 1 is the DHPLC figure of the heavy PCR of TDH, TOXR and TRH3 of VP40 bacterial strain;
Fig. 2 is the electrophorogram of the heavy PCR product of TDH, TOXR and TRH3 of VP40 bacterial strain, wherein M:50bp Marker; The 3 heavy pcr amplification products of 1:TDH, TOXR and TRH; The amplified production of 2:TDH, 196bp; The amplified production of 3:ToxR, 234bp; The amplified production of 4:TRH, 363bp;
Fig. 3 is the DHPLC figure that detects primer sensitivity, is from top to bottom: 10 of VPL4-91 bacterial strain
-1Extent of dilution; 10
-2Extent of dilution; 10
-3Extent of dilution; 10
-4Extent of dilution; 10
-5Extent of dilution; 10
-6Extent of dilution;
Fig. 4 is the electrophorogram that detects primer sensitivity, wherein M:50bpMarker; 10 of 1:VPL4-91 bacterial strain
-1Extent of dilution; 2:10
-2Extent of dilution; 3:10
-3Extent of dilution; 4:10
-4Extent of dilution; 5:10
-5Extent of dilution; 6:10
-6Extent of dilution; 7: blank;
Fig. 5 is the DHPLC figure with Vibrio parahemolyticus of virulence gene, and strain name from top to bottom is: VP40, VPL4-91, AS072, AS073, AS079, ATCC17802;
Fig. 6 is the DHPLC figure of the reference culture of Vibrio parahemolyticus and other Pseudomonas, strain name from top to bottom: VP40, VP1, VP2, VP3, VP4, VP5, VP6, VP7, VP8, VP9, VP10, VP11, VP12, VP13, VP15, VP16, VP18, VP21, VP22, VP23, CMCC53510 artificial tuberculosis yersinia genus, ATCC29544 Enterobacter sakazakii, ATCC25922 intestinal bacteria, ATCC9027 Pseudomonas aeruginosa, CMCC52221 small intestine Ye Ersenshi, CMCC50071 salmonella typhi, CMCC461024 Klebsiella pneumonia, ATCC27562 Vibrio vulnificus, CMCC51334 bacillus ceylonensis A, NICPBP51252 shigella dysenteriae, CMCC26001 streptococcus aureus;
Fig. 7 is that analog sample is used the color atlas that MPCR-DHPLC detects.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
1, materials and methods
1.1 material
1.1.1 bacterium source (as shown in table 1)
Table 1: experimental strain source-information
1.2 key instrument and reagent: Taq enzyme, dNTPs, Buffer, the precious biological ltd in nucleic acid molecular weight mark 100bp ~ 1000bp-Dalian; DNA staining fluid-Ying Wei wound Tianjin company; Triethylamine acetyl salt (TEAA)-U.S. Transgenom ic company; Acetonitrile-U.S. Fisher company; Buffered soln A is the 0.1mol/L TEAA aqueous solution, buffered soln B contain volume(tric)fraction 25% for 0.1mol/L TEAA a acetonitrile solution; API20E-France Biomerieux SA; Triumphant company is encircled in blood agar plate-Guangdong.
1.3 design of primers: the upstream and downstream primer that is directed against the tdh gene: tdhF5 '-gcaccggtcaatgtagaggt-3 ', tdhR:5 '-cagcagaatgaccgtgctta-3 ', amplified fragments size 196bp; Upstream and downstream primer to the trh gene: trhF5 '-aactgaatccccggttaagg-3 ', trh R:5 '-atatgtccattgccgctctc-3 ', amplified fragments size 363bp; Upstream and downstream primer to the toxR gene: toxR F5'-tgatttgcgggtgatttaca-3 ', toxR R:5'-ccgtctttagcgacgacttc-3 ', amplified fragments size 234bp.Entrust the precious biological ltd in Dalian synthetic.
1.4DNA extract: above-mentioned all bacterial strains are all cultivated 10h for 37 ℃ with basic peptone water respectively; Get the 1ml bacteria suspension and move into centrifuge tube, 12000r/min is centrifugal, and 5min removes supernatant, with the floating deposition of 1ml deionized water; 12000r/min is centrifugal, and 3min removes supernatant; Repeat twice, add 200 μ l deionized waters at last and on the nucleic acid extraction appearance, extract DNA, be used for pcr amplification as template DNA.
1.5MPCR-DHPLC the foundation of method:
The optimization of multiplex PCR-DHPLC reaction conditions.
Select for use the VP40 bacterial strain to carry out the optimization of multi-PRC reaction condition; TDH, TOXR and the TRH3 gene because the MPCR reaction is increased simultaneously; The copy number of each gene in reaction is inconsistent, therefore is directed against the ratio of the detection primer of tdh, toxR and trh according to the height adjustment of peak value among the DHPLC figure, confirms that finally the detection primer ratio to TDH:TOXR:TRH is 1:1:3; Result such as Fig. 1, shown in Figure 2, the ratio that therefore is directed against the detection primer of tdh, toxR and trh is 1:1:3.
The MPCR amplified reaction: the reaction TV is 50 μ l, in the 0.2ml reaction tubes, adds 10 * Buffer6 μ l successively, dNTPs5 μ l; Each 1 μ l (20 μ M) of upstream and downstream primer to tdh and toxR gene; To each 3 μ l (20 μ M) of upstream and downstream primer of trh gene, Taq enzyme 1U/ μ l, 0.5 μ l; Distilled water 26.5 μ l, template DNA 2 μ l.Loop parameter is that 95 ℃ of 30s, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations, and 72 ℃ are extended 10min, 4 ℃ of preservations.Pcr amplification product and nucleic acid molecular weight object of reference carry out electrophoresis with 2.2% agarose.Get 5 μ L PCR products simultaneously and carry out the DHPLC analysis, select non-sex change-double-stranded DNA multi-disc section clastotype.50 ℃ of column temperatures, moving phase buffered soln A volume(tric)fraction is 50.2%, buffered soln B volume(tric)fraction is 49.8%, flow velocity 0.9ml/min.
1.6 the detection of multiplex PCR-DHPLC sensitivity: select for use the VPL4-91 bacterial strain to carry out the detection of sensitivity; The VPL4-91 bacterium liquid of drawing 50 μ L adds 225mL APW meat soup and places 36 ± 1 ℃ to cultivate 8 hours, bacterium liquid is carried out 10 times of gradient dilutions after, respectively get 1mL in 3%TSA plate count colony count; And get 1mL bacterium liquid and extract DNA according to the step of DNA extraction test kit; Respectively get 2 μ L as reaction template, carry out the MPCR amplified reaction according to step 1.5, treat that PCR reaction finishes after; Get 10 μ L products and carry out appearance DHPLC on gel electrophoresis and the 5 μ LPCR products, carry out DHPLC according to step 1.5 and analyze.
Its result such as Fig. 3 and shown in Figure 4 can find out that from Fig. 3 and Fig. 4 the minimum detectability of multiplex PCR-DHPLC sensitivity is 10
3Cfu/mL.
1.7MPCR-DHPLC method detects the application of Vibrio parahemolyticus
The aseptic 25g flesh of fish that takes by weighing adds 225mLAPW meat soup; And add 50 μ LVPL4-91 bacterium liquid and place 36 ± 1 ℃ to cultivate after 8 hours, get 1mL meat soup and extract DNA according to the step of DNA extraction test kit, get 2 μ L as reaction template; Carry out the MPCR amplified reaction according to step 1.5; After treating that the PCR reaction finishes, get 10 μ L products and carry out appearance DHPLC on gel electrophoresis and the 5 μ L PCR products, carry out DHPLC according to step 1.5 and analyze.Contrast with traditional detection method simultaneously.
The result of the DHPLC of Vibrio parahemolyticus in the MPCR-DHPLC method detection analog sample is as shown in Figure 7; Can find out by Fig. 7; From analog sample, detected VP; Traditional detection method GB4789.7-2010 " check of food microbiological analysis Vibrio parahemolyticus " also detects VP from analog sample, the result is consistent.
1.8 be directed against the specific detection of the detection primer of tdh, toxR and trh
Select for use the type strain (table 1) of 25 strain Vibrio parahemolyticus (table 1) and 11 other Pseudomonas of strain that the detection primer to tdh, toxR and trh is carried out specific detection; All bacterial strains extract behind the DNA as template DNA according to step 1.4; Carry out the MPCR reaction according to step 1.5 and answer, analyze result such as Fig. 5 and shown in Figure 6 then with DHPLC; Analytical results conforms to the target gene of the bacterial strain of checking shown in the table 2, this shows that detection method specificity of the present invention is high.
Table 2: the target gene of Vibrio parahemolyticus
+: the positive;-: feminine gender.
Claims (6)
1. Vibrio parahemolyticus multiplex PCR-DHPLC detects primer, it is characterized in that, described detection primer is as follows:
To the tdh gene: tdh F5 '-gcaccggtcaatgtagaggt-3 ',
Tdh?R5’-cagcagaatgaccgtgctta-3’；
To the trh gene: trh F5 '-aactgaatccccggttaagg-3 ',
Trh?R5’-atatgtccattgccgctctc-3’；
To the toxR gene: toxR F5'-tgatttgcgggtgatttaca-3',
toxR?R5'-ccgtctttagcgacgacttc-3'。
2. Vibrio parahemolyticus multiplex PCR-DHPLC detection kit comprises the MPCR reaction solution, detects primer and DHPLC reaction reagent, it is characterized in that described detection primer is as follows:
To the tdh gene: tdh F5 '-gcaccggtcaatgtagaggt-3 ',
tdh?R5’-cagcagaatgaccgtgctta-3’；
To the trh gene: trh F5 '-aactgaatccccggttaagg-3 ',
trh?R5’-atatgtccattgccgctctc-3’；
To the toxR gene: toxR F5'-tgatttgcgggtgatttaca-3',
toxR?R5'-ccgtctttagcgacgacttc-3'。
3. the Vibrio parahemolyticus of non-diagnostic purpose multiplex PCR-DHPLC detection method is characterized in that, may further comprise the steps:
(1) sample is increased bacterium and cultivate, the genomic dna that extracts thalline in the enrichment liquid is as template;
(2) use the described detection primer of claim 1 to tdh gene, trh gene and toxR gene; Mix the formation amplification reaction system with the MPCR reaction solution; Carry out the MPCR reaction; After reaction finishes, the PCR reaction product is carried out DHPLC analyzes, according to the DHPLC analytical results judge whether to contain Vibrio parahemolyticus with and the virulence gene somatotype.
4. the Vibrio parahemolyticus of non-diagnostic purpose according to claim 3 multiplex PCR-DHPLC detection method is characterized in that, described amplification reaction system is the system of 50 μ l; Comprise 10 * Buffer, 6 μ l, dNTPs 5 μ l are to each 1 μ l of upstream and downstream primer of tdh and toxR gene; Each 3 μ l of upstream and downstream primer to the trh gene; Taq enzyme 1U/ μ l0.5 μ l, distilled water 26.5 μ l, template DNA 2 μ l.
5. the Vibrio parahemolyticus of non-diagnostic purpose according to claim 3 multiplex PCR-DHPLC detection method; It is characterized in that; Describedly carry out MPCR reaction, its loop parameter is that 95 ℃ of 30s, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations, and 72 ℃ are extended 10min.
6. the Vibrio parahemolyticus of non-diagnostic purpose according to claim 3 multiplex PCR-DHPLC detection method; It is characterized in that it specifically is to get 5 μ L PCR products to carry out the DHPLC analysis that the described DHPLC of carrying out analyzes, and selects non-sex change-double-stranded DNA multi-disc section clastotype; 50 ℃ of column temperatures; Moving phase is that buffered soln A volume(tric)fraction is 50.2%, and buffered soln B volume(tric)fraction is 49.8%, flow velocity 0.9ml/min; Described buffered soln A is the 0.1mol/L TEAA aqueous solution, buffered soln B contain volume(tric)fraction 25% for 0.1mol/L TEAA a acetonitrile solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015190107A1 (en) * | 2014-06-11 | 2015-12-17 | 東洋製罐グループホールディングス株式会社 | Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus |
| CN107988402A (en) * | 2018-01-03 | 2018-05-04 | 北京毅新博创生物科技有限公司 | The kit of Mass Spectrometric Identification vibrio parahemolyticus parting |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006090945A1 (en) * | 2005-02-28 | 2006-08-31 | Samsung Everland Inc. | Primer for detecting food poisoning and method for rapid detection of food born pathogene |
| CN101113475A (en) * | 2007-05-30 | 2008-01-30 | 中国疾病预防控制中心传染病预防控制所 | Primer for detecting pathogenic microorganism and multiple PCR using the same |
| JP2012050362A (en) * | 2010-08-31 | 2012-03-15 | Toyo Seikan Kaisha Ltd | Primer set for multiplex pcr and method for detecting microorganism using the same |
| CN102559889A (en) * | 2011-12-28 | 2012-07-11 | 中国科学院南海海洋研究所 | Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP |
-
2012
- 2012-07-25 CN CN2012102606604A patent/CN102816842A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006090945A1 (en) * | 2005-02-28 | 2006-08-31 | Samsung Everland Inc. | Primer for detecting food poisoning and method for rapid detection of food born pathogene |
| CN101113475A (en) * | 2007-05-30 | 2008-01-30 | 中国疾病预防控制中心传染病预防控制所 | Primer for detecting pathogenic microorganism and multiple PCR using the same |
| JP2012050362A (en) * | 2010-08-31 | 2012-03-15 | Toyo Seikan Kaisha Ltd | Primer set for multiplex pcr and method for detecting microorganism using the same |
| CN102559889A (en) * | 2011-12-28 | 2012-07-11 | 中国科学院南海海洋研究所 | Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP |
Non-Patent Citations (2)
| Title |
|---|
| REKHA D. CHAKRABORTY: "Incidence and Molecular Typing of Vibrio parahaemolyticus from Tiger Shrimp Culture Environments along the Southwest Coast of India", 《FOOD BIOTECHNOLOGY》 * |
| 许龙岩: "MPCR-DHPLC 检测大肠杆菌 O157 研究", 《中国卫生检验杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015190107A1 (en) * | 2014-06-11 | 2015-12-17 | 東洋製罐グループホールディングス株式会社 | Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus |
| CN107988402A (en) * | 2018-01-03 | 2018-05-04 | 北京毅新博创生物科技有限公司 | The kit of Mass Spectrometric Identification vibrio parahemolyticus parting |
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