CN103667498A - Detection method for vibrio parahemolyticus - Google Patents
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Abstract
The invention provides a detection method for vibrio parahemolyticus and relates to vibrio parahemolyticus. Specific primers used for detecting vibrio parahemolyticus are a pair of outer primers, a pair of cross primers and a pair of specificity detection probes; the specific primers are designed according to the conserved region of the specific gene t l h of the vibrio parahemolyticus; the specific gene t l h of the vibrio parahemolyticus is the virulence genes of the coded vibrio parahemolyticus thermolabile hemolysin TLH. The detection method comprises the following steps: DNA extraction of a vibrio parahemolyticus template, validation of outer primers, establishment of a cross primers thermostatic amplification reaction system, establishment of cross primers thermostatic amplification reaction programs, and detection of amplification products. The detection method provided by the invention has the following advantages: the sensitivity is high and is ten times that of the common PCR; the specificity is strong and only vibrio parahemolyticus is detected so that detection results of other pathogenic bacterium are negative; the detection speed is high and the detection time is shortened to 1.5h to remarkably improve the detection efficiency; besides, the detection method is simple and convenient and practical: amplification products can be detected by a disposable test strip in 10 to 30 min.
Description
Technical field
The present invention relates to Vibrio parahemolyticus, especially relate to the detection method of a kind of Vibrio parahemolyticus.
Background technology
Vibrio parahemolyticus claims again halophilic bacterium, is subordinate to the Vibrio of vibrionaceae, is a kind of Gram-negative infecting both domestic animals and human bacterium, is extensively distributed in inshore seawater, marine bottom sediment, sea-food and pickling food.By this microbial food poisoning clinical symptom, mostly be acute enteritis, show as stomachache, diarrhoea, vomiting, be generally 8～20h its latent period, average out to 12h, can fully recover for normal 2～3 days, favourable prognosis, and minority severe patient can be dead not in time because rescuing.Vibrio parahaemolytisus poisoning is relevant with the food that feed contains this bacterium, and eating or eat the sea-food being polluted or the pickling food that do not boil raw is its main routes of transmission.Known important contagium has crab, shrimp, scallop, oyster, clam class, jellyfish, inkfish, salted vegetables, butcher's meat etc.The optimum growth temperature of Vibrio parahemolyticus is 37 ℃, so becomes the high-incidence season of vibrio parahaemolytisus poisoning summer and autumn, and be peak period August.By this microbial food poisoning, leapt to first of China food poisoning in recent years, the foundation of fast and convenient detection method is the effective way of prevention vibrio parahaemolytisus poisoning.
The detection method of the Vibrio parahemolyticus of having set up has traditional method: increase bacterium culture method, maximum possible counting process; Immunological method: enzyme-linked immunosorbent assay, chip method, Radioactive colloidal gold, immunosensor, immunomagnetic beads method; Molecular biology method: ([1] kingdom tinkling of pieces of jade such as conventional PCR, multiplex PCR, quantitative fluorescent PCR, nano PCR, EMA-PCR method, nucleic acid probe, LAMP method, Luan Yuming, Liu Daxiong, Lu Jiahai. the progress of Vibrio parahemolyticus detection method [J]. Chinese Journal of Health Laboratory Technology, 2010,20 (6): 1574-1576).
Cross primer isothermal amplification technology (CPA) is a kind of isothermal amplification technology of novelty, there is higher specificity and sensitivity, because the method just can react under constant temperature, so the requirement to equipment is lower, simple to operate, reaction only needs 60min([2] Fang RD, Li X, Hu L, et al.Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens[J] .Journal of C linical Microbiology, 2009,47 (3): 847-847; [3] Xu GL, Hu L, Zhong HY, et al.Cross priming amplification:mechanism and optimization for isothermal DNA amplification[J] .Scientific Reports, 2012,2:246.doi:10.1038/srep00246).More traditional round pcr has the advantages such as efficient, quick, is especially useful in basic unit Vibrio parahemolyticus is detected.The detection method of the method association colloid gold nucleic acid test strip has been used in detection ([4] Qi Jun such as Salmonellas, enterohemorrhagic Escherichia coli, mycobacterium tuberculosis, pernicious malaria, vibrio cholerae, Shigellae, melon and fruit class pinta bacterium at present, left peak, Liu Guohong, Liu Hongmei, Xu Gaolian, open rosy clouds. cross primer isothermal amplification technique detects Salmonellas [J]. food research exploitation, 2013,34 (2): 67-70; [5] Qi Jun, Yu Zhirui, Zhan Xijing, Huang Jicheng, Li Zhi is intelligent. the application [J] of cross primer isothermal amplification technique in pernicious malaria rapid detection. and Chinese media biology and control magazine, 2013,24 (3): 204-207), be not yet useful on the report that detects Vibrio parahemolyticus.
Summary of the invention
The first object of the present invention is to provide the Auele Specific Primer detecting for Vibrio parahemolyticus.
The second object of the present invention is to provide the detection method of Vibrio parahemolyticus.
The described Auele Specific Primer detecting for Vibrio parahemolyticus is a pair of peripheral primer, pair of cross primer and a pair of specificity detection probe designed according to the conservative region of Vibrio parahemolyticus species specificity gene tlh; Described Vibrio parahemolyticus species specificity gene tlh is the virulence gene of the thermo-labile hemolytic toxin TLH of coding Vibrio parahemolyticus, extensively exists in Vibrio parahemolyticus, and has species specificity;
Described a pair of peripheral primer is:
Primer VPOF:TGCGAAAGTGCTTGAGAT just to the periphery;
Reverse peripheral primer VPOR:GATGAGCGGTTGATGTCC.
Described pair of cross primer is:
Forward cross primer VPIF:TTCTGGCGCAGAAGTTAGGTTCATCAAGGCACAAGC;
Reverse cross primer VPIR:GTTCATCAAGGCACAAGCTTCTGGCGCAGAAGTTAG.
Described a pair of specificity detection probe is:
Forward specificity detection probe VPDF:CAAAGCGCAAGGTTACAACATCA, 3 ' end mark fluorescein isothiocyanate (Fitc);
Reverse specificity detection probe VPDR:GAACAAGGCGTGAGTATCAAACAAC, 5 ' end mark vitamin H (Biotin).
The detection method of described Vibrio parahemolyticus, comprises the following steps:
1) extraction of Vibrio parahemolyticus template DNA
Utilize total DNA of CTAB/NaCl method (with reference to Microbiology Experiment (the 4th edition) chief editor Shen Ping Chen XiangDong) or bacterial genomes DNA extraction test kit (being purchased from TIANGEN Xiamen Tai Jing Bioisystech Co., Ltd) extraction Vibrio parahemolyticus as template;
2) checking of peripheral primer
With a pair of peripheral primer VPOF/VPOR, template is increased, product after amplification carries out plasmid clone and obtains required plasmid, by proof that plasmid is checked order: the fragment of peripheral primer amplification is the conservative region nucleotide sequence of Vibrio parahemolyticus tlh, illustrate that this periphery primer is suitable, can be used for follow-up cross primer constant-temperature amplification method experiment;
3) foundation of cross primer isothermal amplification reactions system
The isothermal reaction system of 20 μ L comprises: peripheral primer 0.1 μ mol/L, cross primer 0.4 μ mol/L, specificity detection probe 0.3 μ mol/L, dNTPs0.4 μ mol/L, MgSO
46mmol/L, 10 * Thermopol buffer2 μ L, Bst DNA Polymerase(U/ μ L) 8U, Betaine0.5mol/L, DNA1 μ L;
4) cross primer constant-temperature amplification program
Question response PCR pipe is positioned over to 63 ℃ of constant-temperature amplification 60min in micro-thermostatted, obtains amplified production;
5) detection of amplified production
Amplified production carries out interpretation by commercial disposable detection of nucleic acids test strip (No. 3) (purchased from Yousida Biological Technology Co., Ltd., Hangzhou), by observing the colour-change of ELISA test strip line and nature controlling line, judges whether reaction occurs; If result is positive, in sample, contain the nucleic acid of detection, there are two red stripes in test strip, and one is positioned at Quality Control district, and one is positioned at detection zone; If result is negative, only have Quality Control district to occur a red stripes, detection zone does not have band.
Compared with prior art, the present invention has following outstanding advantage and practicality:
1. highly sensitive, sensitivity of the present invention is 10 times of regular-PCR;
2. high specificity, the present invention only detects Vibrio parahemolyticus, all negative to other pathogenic microbes detect results;
3. rapid detection, compares with traditional detection method, and the present invention will shorten to 1.5h detection time, obviously improve detection efficiency;
4. simple and practical, amplified production detects by disposable test paper slip, and 10～30min can complete the interpretation of detected result, is especially useful in basic unit's practicality.
Accompanying drawing explanation
Fig. 1 is cross primer constant-temperature amplification product electrophorogram.In Fig. 1, be respectively labeled as M:100bp ladder marker; 1: Vibrio parahemolyticus CPA amplified production; 2: negative control.
Fig. 2 is the nucleic acid test strip detected result figure of cross primer method amplified production.In Fig. 2, be respectively labeled as 1: Vibrio parahemolyticus CPA amplified production; 2: negative control
Fig. 3 is that cross primer constant-temperature amplification detects Vibrio parahemolyticus specificity electrophorogram.In Fig. 3, be respectively labeled as M:100bp ladder marker; 1: negative control; 2: Vibrio parahemolyticus; 3: vibrio cholerae; 4: vibrio alginolyticus; 5: Vibrio vulnificus; 6: streptococcus aureus; 7: intestinal bacteria; 8: Salmonellas.
Fig. 4 is the specificity figure of cross primer constant-temperature amplification bind nucleic acid ELISA test strip Vibrio parahemolyticus.In Fig. 4, be respectively labeled as 1: negative control; 2: Vibrio parahemolyticus; 3: vibrio cholerae; 4: vibrio alginolyticus; 5: Vibrio vulnificus; 6: streptococcus aureus; 7: intestinal bacteria; 8: Salmonellas.
Embodiment
The invention will be further described in connection with accompanying drawing for following examples, but be not used for limiting the scope of the invention.
The design of embodiment 1 primer is with synthetic
According to the conservative region of the thermo-labile hemolytic toxin gene of Vibrio parahemolyticus tlh, designed two pairs of primers and a pair of specificity detection probe, sequence is as follows:
Described a pair of peripheral primer is:
Primer VPOF:TGCGAAAGTGCTTGAGAT just to the periphery;
Reverse peripheral primer VPOR:GATGAGCGGTTGATGTCC.
Described pair of cross primer is:
Forward cross primer VPIF:TTCTGGCGCAGAAGTTAGGTTCATCAAGGCACAAGC;
Reverse cross primer VPIR:GTTCATCAAGGCACAAGCTTCTGGCGCAGAAGTTAG.
Described a pair of specificity detection probe is:
Forward specificity detection probe VPDF:CAAAGCGCAAGGTTACAACATCA, 3 ' end mark fluorescein isothiocyanate (Fitc);
Reverse specificity detection probe VPDR:GAACAAGGCGTGAGTATCAAACAAC, 5 ' end mark vitamin H (Biotin).
The foundation of embodiment 2CPA method
The isothermal reaction system of 20 μ L comprises: peripheral primer 0.1 μ mol/L, cross primer 0.4 μ mol/L, specificity detection probe 0.3 μ mol/L, dNTPs0.4 μ mol/L, MgSO
46mmol/L, 10 * Thermopol buffer2 μ L, Bst DNA Polymerase(U/ μ L) 8U, Betaine0.5mol/L, DNA1 μ L.
CPA response procedures: 63 ℃ of constant-temperature amplification 60min.
The detection of amplified production: get the sample application zone that 8～10 μ L amplified productions are added drop-wise to nucleic acid test strip after amplification finishes.Test strip is put into the microwell plate that contains 100 μ L damping fluids.After 15～30min, can carry out interpretation by the colour developing of test strip.
Embodiment 3CPA-nucleic acid test strip method detection specificity
Respectively the different Vibrio parahemolyticus of 10 strains is detected, result is all positive.For further its specificity of checking, choose respectively vibrio cholerae, Vibrio vulnificus, vibrio alginolyticus, Salmonellas, intestinal bacteria, streptococcus aureus in contrast bacterium test, result shows, contrast bacterium result is all negative.Proof the present invention has good specificity to Vibrio parahemolyticus.
CPA-nucleic acid test strip detects the susceptibility of Vibrio parahemolyticus.By calculating the concentration of pure growth with regard to plate count and it being carried out to 10 times of gradient dilutions, culture after dilution extracts test kit by bacterial genomes and carries out genomic extraction, and the genome after extracting is got to 1 μ L for the susceptibility of CPA reaction detection the method.After testing, lowest detection of the present invention is limited to 5.6 * 10
2cFU/mL, its sensitivity is 10 times of regular-PCR.
Claims (2)
1. the Auele Specific Primer detecting for Vibrio parahemolyticus, is characterized in that the described Auele Specific Primer detecting for Vibrio parahemolyticus is a pair of peripheral primer, pair of cross primer and a pair of specificity detection probe designed according to the conservative region of Vibrio parahemolyticus species specificity gene tlh; Described Vibrio parahemolyticus species specificity gene tlh is the virulence gene of the thermo-labile hemolytic toxin TLH of coding Vibrio parahemolyticus, extensively exists in Vibrio parahemolyticus, and has species specificity;
Described a pair of peripheral primer is:
Primer VPOF:TGCGAAAGTGCTTGAGAT just to the periphery;
Reverse peripheral primer VPOR:GATGAGCGGTTGATGTCC;
Described pair of cross primer is:
Forward cross primer VPIF:TTCTGGCGCAGAAGTTAGGTTCATCAAGGCACAAGC;
Reverse cross primer VPIR:GTTCATCAAGGCACAAGCTTCTGGCGCAGAAGTTAG;
Described a pair of specificity detection probe is:
Forward specificity detection probe VPDF:CAAAGCGCAAGGTTACAACATCA, 3 ' end mark fluorescein isothiocyanate;
Reverse specificity detection probe VPDR:GAACAAGGCGTGAGTATCAAACAAC, 5 ' end mark vitamin H.
2. the detection method of Vibrio parahemolyticus, is characterized in that comprising the following steps:
1) extraction of Vibrio parahemolyticus template DNA
Utilize total DNA of CTAB/NaCl method or bacterial genomes DNA extraction test kit extraction Vibrio parahemolyticus as template;
2) checking of peripheral primer
With a pair of peripheral primer VPOF/VPOR described in claim 1, template is increased, product after amplification carries out plasmid clone and obtains required plasmid, by proof that plasmid is checked order: the fragment of peripheral primer amplification is the conservative region nucleotide sequence of Vibrio parahemolyticus tlh, illustrate that this periphery primer is suitable, can be used for follow-up cross primer constant-temperature amplification method experiment;
3) foundation of cross primer isothermal amplification reactions system
The isothermal reaction system of 20 μ L comprises: peripheral primer 0.1 μ mol/L, cross primer 0.4 μ mol/L, specificity detection probe 0.3 μ mol/L, dNTPs0.4 μ mol/L, MgSO
46mmol/L, 10 * Thermopol buffer2 μ L, Bst DNA Polymerase(U/ μ L) 8U, Betaine0.5mol/L, DNA1 μ L; Described peripheral primer, cross primer, specificity detection probe are peripheral primer described in claim 1, cross primer, specificity detection probe;
4) cross primer constant-temperature amplification program
Question response PCR pipe is positioned over to 63 ℃ of constant-temperature amplification 60min in micro-thermostatted, obtains amplified production;
5) detection of amplified production
Amplified production carries out interpretation No. 3 by commercial disposable detection of nucleic acids test strip, by observing the colour-change of ELISA test strip line and nature controlling line, judges whether reaction occurs; If result is positive, in sample, contain the nucleic acid of detection, there are two red stripes in test strip, and one is positioned at Quality Control district, and one is positioned at detection zone; If result is negative, only have Quality Control district to occur a red stripes, detection zone does not have band.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104328113A (en) * | 2014-10-12 | 2015-02-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence composition and application thereof in Listeria monocytogenes identification |
| CN104388578A (en) * | 2014-12-09 | 2015-03-04 | 中国计量学院 | Method for detecting NOS (nopaline synthase) terminator through crossing primer and dual-probe isothermal amplification |
| CN104561275A (en) * | 2014-12-18 | 2015-04-29 | 浙江省疾病预防控制中心 | Vibrio parahaemolyticus isothermal amplification detection kit and detection method |
| CN105296643A (en) * | 2015-11-20 | 2016-02-03 | 浙江省疾病预防控制中心 | Isothermal amplification detection kit and detection method for duck meat derived component nucleic acid |
| CN105296642A (en) * | 2015-11-20 | 2016-02-03 | 浙江省疾病预防控制中心 | Isothermal amplification detection kit for pork derived component nucleic acid and detection method |
| CN106222274A (en) * | 2016-08-05 | 2016-12-14 | 集美大学 | A kind of method for quick of Martin Hollis Ge Limengte Salmonella |
| CN104328113B (en) * | 2014-10-12 | 2017-01-04 | 中国疾病预防控制中心传染病预防控制所 | One group of nucleotide sequence and the application in Listeria monocytogenes is identified |
| CN106755358A (en) * | 2016-12-05 | 2017-05-31 | 中国疾病预防控制中心传染病预防控制所 | It is intersect the method that amplification combines gold nano bio-sensing detection vibrio parahemolyticus more |
| CN107641659A (en) * | 2017-09-20 | 2018-01-30 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature amplification technique detection vibrio parahaemolytious |
| CN108611432A (en) * | 2018-05-11 | 2018-10-02 | 重庆出入境检验检疫局检验检疫技术中心 | The sandwich DNA hybridization of vibrio parahemolyticus, which quickly detects, uses probe, kit and detection method |
| CN109355405A (en) * | 2018-08-30 | 2019-02-19 | 华南理工大学 | A kind of primer, kit and its method of PSR isothermal amplification detection vibrio parahemolyticus |
| CN110093401A (en) * | 2019-05-10 | 2019-08-06 | 中国农业大学 | A kind of vibrio parahaemolytious detection kit and its detection method |
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| CN104328113B (en) * | 2014-10-12 | 2017-01-04 | 中国疾病预防控制中心传染病预防控制所 | One group of nucleotide sequence and the application in Listeria monocytogenes is identified |
| CN104328113A (en) * | 2014-10-12 | 2015-02-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence composition and application thereof in Listeria monocytogenes identification |
| CN104388578A (en) * | 2014-12-09 | 2015-03-04 | 中国计量学院 | Method for detecting NOS (nopaline synthase) terminator through crossing primer and dual-probe isothermal amplification |
| CN104388578B (en) * | 2014-12-09 | 2016-08-24 | 中国计量学院 | Utilize cross primer and the method for double probe constant-temperature amplification detection NOS terminator |
| CN104561275A (en) * | 2014-12-18 | 2015-04-29 | 浙江省疾病预防控制中心 | Vibrio parahaemolyticus isothermal amplification detection kit and detection method |
| CN105296643A (en) * | 2015-11-20 | 2016-02-03 | 浙江省疾病预防控制中心 | Isothermal amplification detection kit and detection method for duck meat derived component nucleic acid |
| CN105296642A (en) * | 2015-11-20 | 2016-02-03 | 浙江省疾病预防控制中心 | Isothermal amplification detection kit for pork derived component nucleic acid and detection method |
| CN106222274A (en) * | 2016-08-05 | 2016-12-14 | 集美大学 | A kind of method for quick of Martin Hollis Ge Limengte Salmonella |
| CN106222274B (en) * | 2016-08-05 | 2020-01-31 | 集美大学 | quick detection method for hollisgilettia |
| CN106755358A (en) * | 2016-12-05 | 2017-05-31 | 中国疾病预防控制中心传染病预防控制所 | It is intersect the method that amplification combines gold nano bio-sensing detection vibrio parahemolyticus more |
| CN107641659A (en) * | 2017-09-20 | 2018-01-30 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature amplification technique detection vibrio parahaemolytious |
| CN108611432A (en) * | 2018-05-11 | 2018-10-02 | 重庆出入境检验检疫局检验检疫技术中心 | The sandwich DNA hybridization of vibrio parahemolyticus, which quickly detects, uses probe, kit and detection method |
| CN109355405A (en) * | 2018-08-30 | 2019-02-19 | 华南理工大学 | A kind of primer, kit and its method of PSR isothermal amplification detection vibrio parahemolyticus |
| CN110093401A (en) * | 2019-05-10 | 2019-08-06 | 中国农业大学 | A kind of vibrio parahaemolytious detection kit and its detection method |
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