Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP

A detection kit and technology for vibrio hemolyticus, applied in biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc., can solve the problems of single detection content and inability to fully reflect the potential pathogenicity of strains , to achieve the effect of wide detection coverage and improved accuracy

Active Publication Date: 2012-07-11
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in terms of pathogenicity detection of Vibrio parahaemolyticus, the defects of these methods are: the detection content is too single and cannot fully reflect the potential pathogenicity of the strain
Under natural circumstances, the vast majority of Vibrio parahaemolyticus be

Method used

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  • Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP
  • Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP
  • Rapid detection kit and detection method for v.parahaemolyticus multiple virulence factor GeXP

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Embodiment 1

[0059] (1) Extraction of Vibrio parahaemolyticus genomic DNA

[0060] Firstly, the Vibrio parahaemolyticus in the sample was purified and separated.

[0061] ①Add 50 μL 10% (w / v) sterile Chelex-100 solution into the centrifuge tube;

[0062] ②Pick a single colony from the culture plate of Vibrio parahaemolyticus into the above Chelex-100 solution;

[0063] ③ Place the centrifuge tube on a vortex mixer and shake it fully for 10 seconds, take a boiling water bath for 10 minutes, cool to room temperature, and shake it fully for another 10 seconds;

[0064] ④ Centrifuge at 13000r / min for 2min, and the supernatant is the genomic DNA of Vibrio parahaemolyticus, and store it in a -20°C refrigerator for later use.

[0065] (2) Design and synthesis of primers for multigene PCR

[0066] Primers designed for the virulence gene trh, the amplified fragment size is about 147bp:

[0067] trh-F: 5'-AGGTGACACTATAGAATAGCGATTGACCTACCATCCAT-3'

[0068] trh-B: 5'-GTACGACTCACTATAGGGATCAACGATTG...

Embodiment 2

[0143] (1) Preparation of Vibrio parahaemolyticus genomic DNA

[0144] Firstly, the Vibrio parahaemolyticus in the sample was purified and separated.

[0145] ① Add 90 μL 10% (w / v) sterile Chelex-100 solution into the centrifuge tube;

[0146] ②Pick a single colony from the culture plate of Vibrio parahaemolyticus into the above Chelex-100 solution;

[0147] ③ Place the centrifuge tube on a vortex mixer and shake it fully for 15 seconds, take a boiling water bath for 8 minutes, and then shake it fully for 10 seconds after cooling to room temperature;

[0148] ④ Centrifuge at 8000r / min for 20min, and the supernatant is the genomic DNA of Vibrio parahaemolyticus, and store it in a -20°C refrigerator for later use.

[0149] (2) Design and synthesis of primers

[0150] Primers designed for the virulence gene trh, the amplified fragment size is about 147bp:

[0151] trh-F: 5'-AGGTGACACTATAGAATAGCGATTGACCTACCATCCAT-3'

[0152] trh-B: 5'-GTACGACTCACTATAGGGATCAACGATTGCGTTAACTGG-3...

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Abstract

The invention discloses a rapid detection kit and a detection method of a v.parahaemolyticus multiple virulence factor GeXP. The method comprises the following steps of: extracting a genome DNA (Deoxyribose Nucleic Acid) of the v.parahaemolyticus; performing multiple PCR (Polymerase Chain Reaction) amplification on 13 pairs of polygene PCR primers and a pair of general primers by taking the genome DNA as a template to obtain a multiple PCR reaction product; and detecting and analyzing by using a GenomeLab Gexp genetic analysis system. In the invention, a specific primer is designed specific to 13 kinds of known virulence genes of v.parahaemolyticus, and 13 kinds of genes are detected rapidly and efficiently at one time through a single-tube reaction to detect whether a sample contains v.parahaemolyticus which carries virulence genes and has potential pathogenic ability can be known, so that the detection coverage is wide, the accuracy of pathogenicity evaluation of v.parahaemolyticus can be greatly increased, and a more effective detection measure is provided for pathogenicity evaluation of v.parahaemolyticus. The method has important application prospects on the aspects of aquaculture disease detection, epidemiological investigation, pathogenic bacterium detection in foods, aquatic product import and export detection, hospital infection monitoring, and the like.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a multiple virulence factor GeXP rapid detection kit and detection method for detecting pathogenic Vibrio parahaemolyticus (VP.) in clinical examination, public health and food inspection . Background technique: [0002] Vibrio parahaemolyticus (V.Parahaemolyticus) is a halophilic Gram-negative bacterium belonging to the genus Vibrio in the family Vibrio. It is a foodborne pathogenic bacterium mainly derived from seafood such as shrimp and shellfish. The bacteria can not only make seafood pathogenic, but also infect the human body, causing diarrhea, nausea, fever and other typical gastroenteritis reactions and food poisoning. [0003] According to the data released by the National Foodborne Disease Surveillance Network, the distribution of pathogens of microbial food poisoning in my country has changed significantly, especially in coastal provinces, the scale of food po...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 陈偿高磊刘助红胡超群任春华
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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