CN103667498B - The detection method of Vibrio parahemolyticus - Google Patents
The detection method of Vibrio parahemolyticus Download PDFInfo
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Abstract
The detection method of Vibrio parahemolyticus, relates to Vibrio parahemolyticus.Auele Specific Primer for Vibrio parahemolyticus detection is a pair peripheral primer, pair of cross primer and a pair specificity detection probe designed by the conservative region of Vibrio parahemolyticus species-specific genes tlh; Vibrio parahemolyticus species-specific genes tlh is the virulence gene of the thermo-labile hemolytic toxin TLH of coding Vibrio parahemolyticus.Detection method: the extraction of Vibrio parahemolyticus template DNA; The checking of peripheral primer; The foundation of cross primer isothermal amplification reactions system; Cross primer constant-temperature amplification program; The detection of amplified production.Highly sensitive, be 10 times of regular-PCR; High specificity, only detects Vibrio parahemolyticus, is feminine gender to other pathogenic microbes detect results; Rapid detection, detection time shortens to 1.5h, significantly improves detection efficiency; Simple and practical, amplified production is detected by disposable test paper slip, and 10 ~ 30min can complete.
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, also known as 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.Mostly be acute enteritis by this microbial food poisoning clinical symptom, show as stomachache, diarrhoea, vomiting, its latent period is generally 8 ~ 20h, average out to 12h, within normal 2 ~ 3 days, can fully recover, favourable prognosis, and minority severe patient can be dead not in time because rescuing.Vibrio parahaemolytisus poisoning is relevant containing the food of this bacterium with feed, eats or eat the sea-food polluted that do not boil raw or pickling food are its major transmission path.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 DEG C, therefore becomes the high-incidence season of vibrio parahaemolytisus poisoning summer and autumn, and August is peak period.Leapt to first of China poisons by food by this microbial 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: Zengjing Granule 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 Standard PCR, multiplex PCR, quantitative fluorescent PCR, nano PCR, EMA-PCR method, nucleic acid probe, LAMP method, Luan Yuming, Liu Daxiong, Lu Jiahai. the progress [J] of Vibrio parahemolyticus detection method. 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 at a constant temperature, so lower to the requirement of equipment, simple to operate, reaction only needs 60min ([2] FangRD, LiX, HuL, etal.Cross-primingamplificationforrapiddetectionofMycoba cteriumtuberculosisinsputumspecimens [J] .JournalofClinicalMicrobiology, 2009,47 (3): 847-847; [3] XuGL, HuL, ZhongHY, etal.Crossprimingamplification:mechanismandoptimizationf orisothermalDNAamplification [J] .ScientificReports, 2012,2:246.doi:10.1038/srep00246).More traditional round pcr has the advantages such as efficient, quick, is especially useful in basic unit and detects Vibrio parahemolyticus.The detection method of current the method association colloid gold nucleic acid test strip has been used in detection ([4] Qi Jun of such as Salmonellas, enterohemorrhagic Escherichia coli, mycobacterium tuberculosis, pernicious malaria, vibrio cholerae, Shigellae, melon and fruit class pinta bacterium, Zuo Feng, Liu Guohong, Liu Hongmei, Xu Gaolian, Zhang Xia. cross primer isothermal amplification technique detects Salmonellas [J]. and food research is developed, and 2013,34 (2): 67-70; [5] Qi Jun, Yu Zhirui, Zhan Xijing, Huang Jicheng, Li Zhi is intelligent. the application of cross primer isothermal amplification technique in pernicious malaria rapid detection [J]. and Chinese media biology and control magazine, 2013,24 (3): 204-207) report for detecting Vibrio parahemolyticus, is not yet had.
Summary of the invention
The first object of the present invention is to provide the Auele Specific Primer detected for Vibrio parahemolyticus.
The second object of the present invention is to provide the detection method of Vibrio parahemolyticus.
The described Auele Specific Primer for Vibrio parahemolyticus detection is a pair peripheral primer, pair of cross primer and a pair specificity detection probe designed by the conservative region of Vibrio parahemolyticus species-specific genes tlh; Described Vibrio parahemolyticus species-specific genes 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 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 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
CTAB/NaCl method (with reference to Microbiology Experiment (the 4th edition) chief editor Shen Ping Chen XiangDong) or bacterial genomes DNA extraction kit (being purchased from TIANGEN Xiamen Tai Jing Bioisystech Co., Ltd) is utilized to extract the STb gene of Vibrio parahemolyticus as template;
2) checking of peripheral primer
With a pair peripheral primer VPOF/VPOR, template is increased, product after amplification carries out plasmid clone and obtains required plasmid, prove by carrying out order-checking to plasmid: the fragment of peripheral primer amplification is the conservative region nucleotide sequence of Vibrio parahemolyticus tlh, illustrate that this peripheral primer is suitable, can be used for the experiment of follow-up cross primer constant-temperature amplification method;
3) foundation of cross primer isothermal amplification reactions system
The isothermal reaction system of 20 μ L comprises: peripheral primer 0.1 μm of ol/L, cross primer 0.4 μm of ol/L, specificity detection probe 0.3 μm of ol/L, dNTPs0.4 μm of ol/L, MgSO
46mmol/L, 10 × Thermopolbuffer2 μ L, BstDNA polysaccharase (U/ μ L) 8U, trimethyl-glycine 0.5mol/L, DNA1 μ L;
4) cross primer constant-temperature amplification program
Question response PCR pipe is positioned over 63 DEG C of constant-temperature amplification 60min in micro-thermostatted, obtains amplified production;
5) detection of amplified production
Amplified production carries out interpretation by commercial disposable nucleic acid detection test strip (No. 3) (purchased from Yousida Biological Technology Co., Ltd., Hangzhou), judges whether reaction occurs by the colour-change of observing ELISA test strip line and nature controlling line; If result is positive, then the nucleic acid containing detection in sample, there are two red stripes in test strip, one is positioned at quality control region, and one is positioned at detection zone; If result is negative, then only have quality control region 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, is feminine gender to other pathogenic microbes detect results;
3. rapid detection, compared with traditional detection method, the present invention will shorten to 1.5h detection time, significantly improve detection efficiency;
4. simple and practical, amplified production is detected 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, be respectively labeled as, M:100bpladdermarker; 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 figure 3, be respectively labeled as, M:100bpladdermarker; 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 the diagram, 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
Following examples will the invention will be further described by reference to the accompanying drawings, but be not used for limiting the scope of the invention.
The Design and synthesis of embodiment 1 primer
Conservative region according to Vibrio parahemolyticus thermo-labile hemolytic toxin gene tlh devises two pairs of primers and a pair specificity detection probe, and sequence is as follows:
Described a pair 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 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 μm of ol/L, cross primer 0.4 μm of ol/L, specificity detection probe 0.3 μm of ol/L, dNTPs0.4 μm of ol/L, MgSO
46mmol/L, 10 × Thermopolbuffer2 μ L, BstDNA polysaccharase (U/ μ L) 8U, trimethyl-glycine 0.5mol/L, DNA1 μ L.
CPA response procedures: 63 DEG C 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 terminates.Test strip is put into the microwell plate containing 100 μ L damping fluids.Interpretation is carried out in colour developing namely by test strip after 15 ~ 30min.
Embodiment 3CPA-nucleic acid test strip method detection specificity
Vibrio parahemolyticus different to 10 strains respectively detects, and result is the positive.In order to verify its specificity further, choose respectively vibrio cholerae, Vibrio vulnificus, vibrio alginolyticus, Salmonellas, intestinal bacteria, streptococcus aureus in contrast bacterium test, result shows, and contrast bacterium result is feminine gender.Prove that 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 carrying out 10 times of gradient dilutions to it, culture bacterial genomes after dilution is extracted test kit and is carried out genomic extraction, the genome after extraction is got the susceptibility that 1 μ L is used for 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 (1)
1. the detection method of Vibrio parahemolyticus, described method is used for the detection to inshore seawater, marine bottom sediment, sea-food and pickling food, be not used in the medical diagnosis on disease to lived human body and animal body, it is characterized in that described detection method adopts the Auele Specific Primer detected for Vibrio parahemolyticus, the described Auele Specific Primer for Vibrio parahemolyticus detection is a pair peripheral primer, pair of cross primer and a pair specificity detection probe designed by the conservative region of Vibrio parahemolyticus species-specific genes tlh; Described Vibrio parahemolyticus species-specific genes 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 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 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;
Described detection method, comprises the following steps:
1) extraction of Vibrio parahemolyticus template DNA
CTAB/NaCl method or bacterial genomes DNA extraction kit is utilized to extract the STb gene of Vibrio parahemolyticus as template;
2) checking of peripheral primer
With a pair peripheral primer VPOF/VPOR, template is increased, product after amplification carries out plasmid clone and obtains required plasmid, prove by carrying out order-checking to plasmid: the fragment of peripheral primer amplification is the conservative region nucleotide sequence of Vibrio parahemolyticus tlh, illustrate that this peripheral primer is suitable, can be used for the experiment of follow-up cross primer constant-temperature amplification method;
3) foundation of cross primer isothermal amplification reactions system
The isothermal reaction system of 20 μ L comprises: peripheral primer 0.1 μm of ol/L, cross primer 0.4 μm of ol/L, specificity detection probe 0.3 μm of ol/L, dNTPs0.4 μm of ol/L, MgSO
46mmol/L, 10 × Thermopolbuffer2 μ L, BstDNA polysaccharase 8U/ μ L, trimethyl-glycine 0.5mol/L, DNA1 μ L;
4) cross primer constant-temperature amplification program
Question response PCR pipe is positioned over 63 DEG C of constant-temperature amplification 60min in micro-thermostatted, obtains amplified production;
5) detection of amplified production
Amplified production carries out interpretation by commercial No. 3, disposable nucleic acid detection test strip, judges whether reaction occurs by the colour-change of observing ELISA test strip line and nature controlling line; If result is positive, then the nucleic acid containing detection in sample, there are two red stripes in test strip, one is positioned at quality control region, and one is positioned at detection zone; If result is negative, then only have quality control region to occur a red stripes, detection zone does not have band.
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| 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 |
| CN106222274B (en) * | 2016-08-05 | 2020-01-31 | 集美大学 | quick detection method for hollisgilettia |
| CN106755358B (en) * | 2016-12-05 | 2020-09-01 | 中国疾病预防控制中心传染病预防控制所 | Method for detecting vibrio parahaemolyticus by combining multi-cross amplification with gold nano biosensing |
| 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 |
| CN109355405B (en) * | 2018-08-30 | 2022-10-25 | 华南理工大学 | Primer, kit and method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction |
| 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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