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Multiplex quantitative PCR (polymerase chain reaction) detection kit for vibrio parahaemolyticus and detection method

A hemolytic vibrio and multiple technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of long cycle, inability to achieve standardized judgment, complicated operation, etc.

Inactive Publication Date: 2012-07-25
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
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Problems solved by technology

The traditional identification methods of Vibrio parahaemolyticus mainly include isolation and culture, biochemical tests, serotyping, etc., because of the complicated operation and long cycle, they can no longer meet the needs of current practical work (on-site diagnosis of infectious diseases, analysis of epidemic trends, etc.) , and for the diagnosis of toxins, classical cytology methods are often used, which cannot achieve standardized determination

Method used

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  • Multiplex quantitative PCR (polymerase chain reaction) detection kit for vibrio parahaemolyticus and detection method
  • Multiplex quantitative PCR (polymerase chain reaction) detection kit for vibrio parahaemolyticus and detection method
  • Multiplex quantitative PCR (polymerase chain reaction) detection kit for vibrio parahaemolyticus and detection method

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Experimental program
Comparison scheme
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Embodiment 1

[0039] Embodiment 1: the acquisition of standard product

[0040] 1. Materials:

[0041] The pGEM-T-Easy cloning system, PCR-related reagents and Taq DNA polymerase were purchased from Promega Company of the United States, 377 sequencer (ABI Company), Bio-Radi cycler PCR instrument (Bio-Rad Company), ABI7500 fast quantitative PCR instrument ( ABI Corporation).

[0042] 2. Primer and probe design and synthesis:

[0043]Vibrio parahaemolyticus tdh (thermostable direct hemolysin) gene (Genbank registration number is GU971653), tlh (thermolabile hemolysin) gene (Genbank registration number is AY829372), toxR (toxin expression regulatory protein) gene (Genbank The registration number is JN188468) and trh (thermoresistance-related hemolysin) gene (Genbank registration number is DQ359749) as templates, use Primer Express TM (V3.0, U.S. ABI company) software analysis TaqMan primer and probe site, select from best combination. The primers and probes were synthesized and purified by...

Embodiment 2

[0057] Example 2: Establishment of multiplex fluorescent quantitative PCR method for detection of four related toxin genes of Vibrio parahaemolyticus

[0058] 1. Plasmid DNA and other bacterial DNA extraction:

[0059] Use genomic DNA extraction reagent to extract bacterial genomic DNA, and use plasmid DNA extraction kit to extract positive plasmid DNA. Take 1.0 μL (50ng / μL) as template respectively, and use upstream and downstream primers for detection on ABI7500 fast quantitative PCR instrument (ABI company) Perform PCR amplification.

[0060] The composition of the PCR reaction solution is as follows:

[0061]

[0062]

[0063] Make up to 50 μL with deionized water.

[0064] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 40 cycles of amplification at 95°C for 15 seconds, and 45 seconds at 60°C, and finally placed at 4°C. Fluorescence acquisition was performed at the annealing temperature of each cycle.

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Abstract

The invention provides a multiplex quantitative PCR (polymerase chain reaction detection kit for vibrio parahaemolyticus toxin gene and a detection method. The kit mainly comprises specific primers, probes and PCR reaction reagent, wherein the specific primers and the probes consist of specific primers and probes of vibrio parahaemolyticus thermostable direct hemolysin gene (tdh), thermolabile hemomysin gene (tlh), toxin expression regulating protein gene (toxR) and thermostable related hemolysin gene (trh). The invention provides the quick, sensitive and specific multiplex fluorescent quantitative PCR detection kit and the detection method aiming at the vibrio parahaemolyticus toxin gene, and provides basis for controlling food poisoning caused by the vibrio parahaemolyticus in time and early diagnosis of the food poisoning caused by the vibrio parahaemolyticus.

Description

(1) Technical field [0001] The invention relates to a multiple quantitative PCR detection kit and a detection method for vibrio parahaemolyticus toxin gene. (2) Background technology [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, Vp) is a Gram-negative halophilic Vibrio and one of the more important pathogenic bacteria in the Vibrio genus. In its sediments, it is the main pathogenic bacteria of food poisoning and acute diarrhea in summer and autumn in coastal areas, which can cause acute gastrointestinal symptoms. At present, the incidence of Vibrio parahaemolyticus ranks first among various microbial food-borne diseases, and is significantly higher than that of Salmonella food poisoning, which ranks second, especially in coastal areas. It is reported that Vibrio parahaemolyticus carries a variety of toxin genes, and the toxin proteins expressed by these genes are the main cause of food poisoning. The traditional identification methods of Vibrio parahaemolyticu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
CPCY02A50/30
Inventor 叶菊莲金大智罗芸张政王复甦
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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