CN102732599A - Method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in seawater sample by duplex polymerase chain reaction - Google Patents
Method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in seawater sample by duplex polymerase chain reaction Download PDFInfo
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- CN102732599A CN102732599A CN2011100908198A CN201110090819A CN102732599A CN 102732599 A CN102732599 A CN 102732599A CN 2011100908198 A CN2011100908198 A CN 2011100908198A CN 201110090819 A CN201110090819 A CN 201110090819A CN 102732599 A CN102732599 A CN 102732599A
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- vibrio
- vibrio parahaemolyticus
- vibrio vulnificus
- vulnificus
- parahaemolyticus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
Belonging to the field of water pathogenic microorganism detection in environmental science, the invention relates to a method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in a seawater sample by duplex polymerase chain reaction. The method comprises: designing two pairs of primers with a generally identical annealing temperature based on the conserved sequences of Vibrio vulnificus and Vibrio parahaemolyticus; adding the two pairs of primers and DNA templates of the two bacteria to a same reaction system, conducting the circulation of denaturation, annealing, and extending dozens of times to make target genes amplified by dozens or even hundreds of times, thus being able to easily detect the presence of the target genes and detect the presence of Vibrio vulnificus or Vibrio parahaemolyticus in the seawater sample simultaneously. Compared with traditional methods, the method of the invention is characterized by strong specificity, high sensitivity, simple and efficient operation, and also time, labor as well as cost saving. In addition, the two bacteria detected by the method provided in the invention are very common pathogenic microorganisms in the ocean, so that the method is endowed with practical significance and application value, and is suitable for promotion.
Description
Technical field:
The invention belongs to environmental science water body pathogenic micro-organism detection range; Be to use polymerase chain reaction (full name Polymerase Chain Reaction; Be called for short PCR) technology; With micro-target DNA fragment amplification, Vibrio vulnificus in the rapid detection water sample and Vibrio parahaemolyticus are estimated the also pollution condition of these two kinds of pathogenic bacterium of early warning ocean environment.
Background technology:
Vibrio vulnificus (Vibrio vulnificus) is a kind of Gram-negative vibrios, can infect human body through wound, the antihygienic sea-food of diet, causes diarrhea and vomiting, tachycardia, myositis, muscular death, causes septicemia, even threat to life.Vibrio parahemolyticus (Vibrio parahaemolyticus) is claimed halophilic bacterium again, also is Gram-negative bacteria; Can secrete heating property hemolysin and similar hemolytic poison; Have hemolytic activity, enterotoxin and lethal effect, can cause human abdominal pain diarrhea, vomiting, mycogastritis, even internal organ extravasated blood.2003 and 2005,, in the illness outbreak of mikrobe property food source, headed by the outbreak of disease that all causes with Vibrio parahaemolyticus, be respectively 21.0% and 40.1% according to the monitoring materials analysis of China food origin disease outburst.These two kinds of pathogenic bacterium all extensively distribute in the ocean, also contain this two kinds of pathogenic micro-organisms in many sea-foods, threaten human life and health safety.
Biological method with conventional detects sample, relatively wastes time and energy, and generally needs 5-7 days.Along with the development of PCR (polymerase chain reaction) technology,, high specificity easy and simple to handle, highly sensitive characteristic because of it, existing scholar has carried out round pcr is applied to the research of sample detection aspect, detects pathogenic micro-organism quickly and accurately in the hope of reaching.Double PCR adds two pairs of primers in same reaction system on conventional PCR basis, amplify two segment DNA fragments simultaneously, has saved time and cost so more.The Vibrio vulnificus and the Vibrio parahaemolyticus of therefore setting up in the dual-PCR method rapid detection seawater sample have important and practical meanings and using value, are suitable for promoting.
Summary of the invention:
The purpose of this invention is to provide a kind of detection method that can detect Vibrio vulnificus and Vibrio parahaemolyticus in the seawater sample quickly and accurately.The key of double PCR is the two pairs of primers that annealing temperature is identical of conserved sequence design to two kinds of bacteriums, only needs 6-8 hour detection time.
The present invention realizes through following technical scheme:
1, it is following to be used for two pairs of primer nucleotide sequences of double PCR:
Vv-F：ATGTTTATGGTGAGAACGGT
Vv-R：GGGGTTACTTGAACATTACG
Vp-F：AAAGCGGATTATGCAGAAGCACTG
Vp-R：GCTACTTTCTAGCATTTTCTCTGC
2, the preparation of dna profiling
Vibrio vulnificus and Vibrio parahaemolyticus are inoculated into respectively in the sterilized LB liquid nutrient medium; Vibrating, (Vibrio vulnificus was in 28 ℃ in 18 hours; Vibrio parahaemolyticus is in 37 ℃), the bacterium liquid of gained extracts the genomic dna of two kinds of bacteriums with the certain multiple of filtering aseptic seawater dilution with test kit.
3, double PCR reaction
(1) total liquid volume of reaction is 25 μ l, and system is formed as follows:
| Composition | Volume (μ l) | Final concentration |
| 10×Buffer | 2.5 | 1×Buffer |
| MgCl 2(25mM) | 2.5 | 2.5mM |
| 4×dNTPs(10mM) | 2 | 0.8mM |
| Tap enzyme (5U/ μ l) | 0.25 | 0.05U |
| Vv-F(10mM) | 1 | 0.4mM |
| Vv-R(10mM) | 1 | 0.4mM |
| Vp-F(10mM) | 1 | 0.4mM |
| Vp-R(10mM) | 1 | 0.4mM |
| The Vibrio vulnificus dna profiling | 2 | |
| The Vibrio parahaemolyticus dna profiling | 2 | |
| Distilled water | 9.75 |
(2) the double PCR amplification program is following:
1. 94 ℃ of preparatory sex change are 5 minutes;
2. three temperature circulations are 35 times:
94 ℃ of sex change 40 seconds;
Annealed 1 minute for 58 ℃;
74 ℃ were extended 1 minute;
3. 74 ℃ were extended 10 minutes eventually
(3) amplified production detects
The double PCR product carries out agarose gel electrophoresis, contains the EB (being used for dna fragmentation dyeing) of 0.5mg/ml in 2% the glue, under gel imaging system, observes, and the expanding fragment length of Vibrio vulnificus and Vibrio parahaemolyticus is respectively 300bp and 450bp.
Advantage of the present invention is following:
1. save time and cost.Conventional biological method needs 5-7 days, though the regular-PCR method also can be accomplished detection within one day, need carry out twice experimental implementation.The present invention unites two into one twice operation, carries out one group of experiment and can obtain the detected result to two kinds of pathogenic bacterias.
2. have very high sensitivity and specificity.The present invention be directed to the conserved sequence designed primer of two kinds of pathogenic bacterium, can both reach 2 * 10 the detectabilities of two kinds of pathogenic bacterium
3About CFU/mL, very sensitive.
3. detecting operation is simple, need not use complex instrument, only need carry out simple training to operator and get final product; Agents useful for same is all nontoxic, and preparation is convenient.
4. be widely used.Core of the present invention---double PCR technology not only can be applied in the detection of Vibrio vulnificus and Vibrio parahaemolyticus in the seawater sample; For Vibrio vulnificus in other environmental samples and the sea-food and Vibrio parahaemolyticus, all can after extracting genome, carry out rapid detection.
Description of drawings:
The double PCR amplified production detected result of sample.
Embodiment:
1. extract the bacterial genomes DNA in the water sample according to kit method, as template.
2. according to the following table preparing reaction solution
| Composition | Volume (μ l) | Final concentration |
| 10×Buffer | 2.5 | 1×Buffer |
| MgCl 2(25mM) | 2.5 | 2.5mM |
| 4×dNTPs(10mM) | 2 | 0.8mM |
| Tap enzyme (5U/ μ l) | 0.25 | 0.05U |
| Vv-F(10mM) | 1 | 0.4mM |
| Vv-R(10mM) | 1 | 0.4mM |
| Vp-F(10mM) | 1 | 0.4mM |
| Vp-R(10mM) | 1 | 0.4mM |
| The DNA of bacteria template | 2-5 | |
| Distilled water | 6.75-9.75 |
The TV of solution is 25 μ l, and the DNA of bacteria template is looked bacterial concentration in the liquid and decided, and generally is about 2-5 μ l, adds distilled water at last and is settled to 25 μ l.
3. three temperature circulations are increased, and program is following:
1. 94 ℃ of preparatory sex change are 5 minutes;
2. three temperature circulations are 35 times:
94 ℃ of sex change 40 seconds;
Annealed 1 minute for 58 ℃;
74 ℃ were extended 1 minute;
3. 74 ℃ were extended 10 minutes eventually
4. amplified production carries out the agarose gel electrophoresis detection
Contain the EB (being used for dna fragmentation dyeing) of 0.5mg/ml in 2% the glue, under gel imaging system, observe, the expanding fragment length of Vibrio vulnificus and Vibrio parahaemolyticus is respectively 300bp and 450bp.See accompanying drawing 1.
In the accompanying drawing, swimming lane 1 is marker, and the length of five bands is followed successively by 100bp, 400bp, 800bp, 1500bp, 2000bp from bottom to top;
Swimming lane 2 is amplifications of Vibrio vulnificus substance PCR, and band length is 300bp;
Swimming lane 3 is amplifications of Vibrio parahaemolyticus substance PCR, and band length is 450bp;
Swimming lane 4 is negative controls;
Road, swimming lane 5 roads-8 is the double PCR amplification of different templates concentration, and template concentrations reduces by 10 times successively.Sequence list:
Vibrio vulnificus primer upper reaches Vv-F:ATGTTTATGGTGAGAACGGT, 20bp
Vibrio vulnificus primer downstream Vv-R:GGGGTTACTTGAACATTACG, 20bp
Vibrio parahaemolyticus primer upper reaches Vp-F:AAAGCGGATTATGCAGAAGCACTG, 24bp
Vibrio parahaemolyticus primer downstream Vp-R:GCTACTTTCTAGCATTTTCTCTGC, 24bp
Claims (2)
1. dual polymerase chain reaction method detects Vibrio vulnificus and the Vibrio parahaemolyticus in the seawater sample, it is characterized in that the primer of these two kinds of pathogenic bacterium is joined in the same system, and the present invention realizes through following technical scheme:
1) it is following, to be used for two pairs of primer nucleotide sequences of double PCR:
Vv-F：ATGTTTATGGTGAGAACGGT
Vv-R：GGGGTTACTTGAACATTACG
Vp-F：AAAGCGGATTATGCAGAAGCACTG
Vp-R：GCTACTTTCTAGCATTTTCTCTGC
2), the preparation of dna profiling
Vibrio vulnificus and Vibrio parahaemolyticus are inoculated into respectively in the sterilized LB liquid nutrient medium; Vibrating, (Vibrio vulnificus was in 28 ℃ in 18 hours; Vibrio parahaemolyticus is in 37 ℃), the bacterium liquid of gained extracts the genomic dna of two kinds of bacteriums with the certain multiple of filtering aseptic seawater dilution with test kit.
3), double PCR reaction
(1) total liquid volume of reaction is 25 μ l, and system is formed as follows:
(2) the double PCR amplification program is following:
1. 94 ℃ of preparatory sex change are 5 minutes;
2. three temperature circulations are 35 times:
94 ℃ of sex change 40 seconds;
Annealed 1 minute for 58 ℃;
74 ℃ were extended 1 minute;
3. 74 ℃ were extended 10 minutes eventually
(3) amplified production detects
The double PCR product carries out agarose gel electrophoresis, contains the EB (being used for dna fragmentation dyeing) of 0.5mg/ml in 2% the glue, under gel imaging system, observes, and the expanding fragment length of Vibrio vulnificus and Vibrio parahaemolyticus is respectively 300bp and 450bp.
2. the described dual polymerase chain reaction method of claim 1 detects Vibrio vulnificus and Vibrio parahaemolyticus in the seawater sample, can be used for detecting Vibrio vulnificus and the Vibrio parahaemolyticus in seawater sample and other water samples.
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| CN2011100908198A CN102732599A (en) | 2011-04-12 | 2011-04-12 | Method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in seawater sample by duplex polymerase chain reaction |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981275A (en) * | 2014-05-30 | 2014-08-13 | 浙江万里学院 | Multi-PCR (Polymerase Chain Reaction) primers used for simultaneously detecting four types of pathogenic bacteria in ocean and design method thereof |
| CN104017873A (en) * | 2014-05-30 | 2014-09-03 | 浙江万里学院 | A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof |
| CN105219845A (en) * | 2015-07-28 | 2016-01-06 | 青岛农业大学 | The dual LAMP method of Vibrio parahaemolyticus and Vibrio vulnificus can be detected simultaneously |
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| CN1560274A (en) * | 2004-02-26 | 2005-01-05 | 中山大学 | Reagent case for diagnosing gene of pathogenic bacterial and para hematolysis vibrion of marine water product animal and hunman and testing method thereof |
| CN101113473A (en) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique |
| CN101633952A (en) * | 2009-03-20 | 2010-01-27 | 邵碧英 | Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof |
| CN101818189A (en) * | 2010-04-16 | 2010-09-01 | 中国水产科学研究院珠江水产研究所 | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc |
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2011
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Patent Citations (4)
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| CN1560274A (en) * | 2004-02-26 | 2005-01-05 | 中山大学 | Reagent case for diagnosing gene of pathogenic bacterial and para hematolysis vibrion of marine water product animal and hunman and testing method thereof |
| CN101113473A (en) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique |
| CN101633952A (en) * | 2009-03-20 | 2010-01-27 | 邵碧英 | Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof |
| CN101818189A (en) * | 2010-04-16 | 2010-09-01 | 中国水产科学研究院珠江水产研究所 | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981275A (en) * | 2014-05-30 | 2014-08-13 | 浙江万里学院 | Multi-PCR (Polymerase Chain Reaction) primers used for simultaneously detecting four types of pathogenic bacteria in ocean and design method thereof |
| CN104017873A (en) * | 2014-05-30 | 2014-09-03 | 浙江万里学院 | A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof |
| CN104017873B (en) * | 2014-05-30 | 2017-05-10 | 浙江万里学院 | A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof |
| CN105219845A (en) * | 2015-07-28 | 2016-01-06 | 青岛农业大学 | The dual LAMP method of Vibrio parahaemolyticus and Vibrio vulnificus can be detected simultaneously |
| CN105219845B (en) * | 2015-07-28 | 2018-10-30 | 青岛农业大学 | The dual LAMP method of vibrio parahaemolytious and Vibrio vulnificus can be detected simultaneously |
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Application publication date: 20121017 |