Primers and reagent kit for rapidly detecting specific vibrio parahemolyticus virulence plasmid pVA1 on site

A hemolytic Vibrio, specific technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of rapid monitoring and detection application of unfavorable farmers, time-consuming, false positives, etc. , to achieve the effect of intuitive detection results, accurate method and high accuracy

Pending Publication Date: 2016-04-20
海峡两岸农产品检验检疫技术厦门中心
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Problems solved by technology

[0006] At present, the most accurate means of detecting this specific Vibrio parahaemolyticus is the method of PCR detection of pVA1 plasmid established in 2013. This conventional PCR detection method requires a series of PCR instruments, electrophoresis instruments, ultraviolet detectors, computers, etc. Professional detection equipment; at the same time, the detection process needs to draw

Method used

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  • Primers and reagent kit for rapidly detecting specific vibrio parahemolyticus virulence plasmid pVA1 on site
  • Primers and reagent kit for rapidly detecting specific vibrio parahemolyticus virulence plasmid pVA1 on site
  • Primers and reagent kit for rapidly detecting specific vibrio parahemolyticus virulence plasmid pVA1 on site

Examples

Experimental program
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Embodiment

[0020] Example: Identification of Vibrio parahaemolyticus virulence plasmid pVA1

[0021] 1) Isolation and identification of specific Vibrio parahaemolyticus

[0022] The hepatopancreas of prawn seedlings infected by Vibrio parahaemolyticus were collected from the Zhangpu prawn farm, and the purified Vibrio parahaemolyticus was isolated and purified according to the conventional isolation and identification method of Vibrio parahaemolyticus. The gene fragment of plasmid pVA1 was amplified by PCR with primers AP3F: 5'-ATGAGTAACAATATAAAACATGAAAC-3' (SEQ ID NO: 1) and AP3R: 5'-GTGGTAATAGATTGTACAGAA-3' (SEQ ID NO: 2) (purified Vibrio parahaemolyticus was used as a template) After PCR product sequencing and Blast comparison, it was determined that the isolated Vibrio parahaemolyticus was a specific Vibrio parahaemolyticus strain carrying the plasmid pVA1, and the strain was preserved for future use.

[0023] 2) DNA extraction

[0024] Take the liquid culture of the above-mentione...

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Abstract

The invention discloses primers and a reagent kit for rapidly detecting the specific vibrio parahemolyticus virulence plasmid pVA1 on site. The sequences of the primers are shown as SE ID NO:3-6. By the adoption of the primers, the specific vibrio parahemolyticus virulence plasmid pVA1 can be easily, conveniently, rapidly and visually detected, accuracy is high, specificity is good, detection time is short, and operation is easy and convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer and a kit for on-site rapid detection of specific Vibrio parahaemolyticus virulence plasmid pVA1. Background technique [0002] Vibrio parahemolyticus (Vibrioparahemolyticus) is a Gram-negative bacillus with arc-shaped, rod-shaped, filamentous and other shapes without spores. At present, the PCR methods used to detect the bacteria mainly include arbitrary primer PCR (arbitrarilyprimedPCR, Ap-PCR), conventional PCR, multiplex PCR (multiplex-PCR), real-time PCR (Real-timePCR). [0003] The thermolabile hemolytic toxin tlh gene was discovered by Hatsumi et al. in 1985. Both clinical isolates and environmental isolates have tlh gene, which is species-specific. Therefore, its coding gene tlh gene is often used as the target gene for detection of Vibrio parahaemolyticus. However not all V. parahaemolyticus are equally pathogenic. Thermostable direct hemolytic toxin TDH and ther...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107Y02A50/30
Inventor 郭书林陈信忠孔繁德龚艳清杨俊萍
Owner 海峡两岸农产品检验检疫技术厦门中心
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