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Salmonella, Listeria monocytogenes, bacillus coli multiple PCR rapid detection kit and uses thereof

A detection kit and Salmonella technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome inspection methods, inconvenient import and export, and low specificity, and achieve high sensitivity and operation Simple, well-specific effects

Inactive Publication Date: 2008-05-07
CHINA AGRI UNIV
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Problems solved by technology

The traditional national and industry standards for Salmonella and Listeria monocytogenes mostly use traditional plate culture or enzyme-linked immunoreaction methods, and it takes at least 7 to 10 days to obtain the test results, and different test procedures are used for the operation , method, and report separately, the inspection method is cumbersome, time-consuming and labor-intensive, and has low specificity, which brings great inconvenience to the import and export of food

Method used

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  • Salmonella, Listeria monocytogenes, bacillus coli multiple PCR rapid detection kit and uses thereof

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Embodiment Construction

[0031] specific implementation plan

[0032] 1. Collect samples to be tested and cultivate pathogenic bacteria

[0033] Collect the samples to be tested, pay attention to avoid cross-contamination before and after collection, inoculate them in broth culture medium at 37°C for 18 hours (to cultivate Salmonella), and culture them in BHI (purchased from Difo, USA, to cultivate Listeria) overnight. LB medium (cultivation of Escherichia coli) was cultured overnight at 37°C.

[0034] And the cultured bacterial liquid is carried out as follows:

[0035] Escherichia coli and Salmonella: take 0.5mL bacterial culture solution, put it in an Eppendorf tube, centrifuge at 2000rpm for 20min, wash twice with sterilized distilled water, and finally suspend with 1mL distilled water, boil in a water bath, centrifuge at 8000rpm for 15min, take the supernatant, and obtain Template solutions for E. coli and Salmonella.

[0036] Listeria: Add 250ul of overnight culture into an Eppendorf tube, ce...

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Abstract

The invention discloses a method simply and rapidly detecting Salmonella, Listeria monocytogenes producing monocyte, and escherichia coli simultaneously and a detection reagent kit invented based on the method. Firstly the DNA templates of the three microorganisms are extracted through hot dehiscing (Salmonella, escherichia coli) and enzymolysis method (Listeria monocytogenes), then the specific primers according to salmonella, Listeria monocytogenes producing monocyte, and escherichia coli are designed respectively, the practical amplification fragments are salmonella 320bp, Listeria monocytogenes producing monocyte 440bp, and escherichia coli 706bp. The invention has the advantages of celerity and nicety compared with the bacteria separation, identification of classical method and serological typing test method, strong specificity and high sensitivity compared with the generic multiplex PCR method, and the ability to detect and identify the three pathogens simultaneously.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and discloses a multiple PCR method and a detection kit for simultaneously detecting three pathogenic bacteria of Salmonella, Listeria monocytogenes and Escherichia coli. Background technique [0002] Salmonella and Listeria monocytogenes are important zoonotic pathogens, and are the two most important foodborne pathogens listed by the World Health Organization (WHO). Salmonella can not only cause animal diseases, but also cause typhoid fever, paratyphoid fever, sepsis, gastroenteritis and food poisoning in humans. The food poisoning cases caused by this are the first or second in food poisoning. Listeria monocytogenes is a food-borne pathogen that can cause diseases such as abortion, sepsis and meningitis in animals and humans. Newborns, the elderly, pregnant women and immunocompromised patients are infected by Listeria monocytogenes high-risk groups. In developed countries, the inci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12Q1/10
CPCY02A50/30
Inventor 罗云波许文涛黄昆仑白卫滨元延芳
Owner CHINA AGRI UNIV
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