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Primers and method for simultaneously detecting various pathogenic bacteria

A technology of pathogenic bacteria and primer pairs, applied in the field of primers and detection of multiple pathogenic bacteria at the same time, can solve the problems of the food safety management system and the legal and regulatory system to be improved, incompatibility, etc., and avoid false positive results. , high sensitivity and good specificity

Active Publication Date: 2013-11-13
广州金苗动保科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(2) There are still potential safety hazards in the supply of inputs, the environment of agricultural production areas, the epidemic prevention system, the production, processing and sales of agricultural products, etc.
(4) The food safety standard system, inspection and testing system, certification and accreditation system, etc. still have obvious incompatibility, and the food safety management system and laws and regulations system need to be improved
[0005] There is currently no set of detection methods that can detect the presence of the above-mentioned pathogenic bacteria at the same time

Method used

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  • Primers and method for simultaneously detecting various pathogenic bacteria
  • Primers and method for simultaneously detecting various pathogenic bacteria
  • Primers and method for simultaneously detecting various pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Design and Screening of Five Kinds of Pathogenic Bacteria Detection Primers

[0074] 1.1 Experimental materials

[0075] Main tools Enzymes, biochemical reagents and kits:

[0076] LB broth, various biochemical tubes, Listeri enrichment solution and other reagents were purchased from Guangzhou Huankai Biological Reagent Co., Ltd.

[0077] Taq DNA polymerase (Fermentas), lysozyme, bacterial genomic DNA extraction kit (Tiangen), ordinary agarose gel, DNA recovery kit (Tiangen), pGM-T19 ligation kit (including vector, ligase ) was purchased from Guangzhou Baotech Biotechnology Co., Ltd., plasmid DNA mini-extraction kit (Tiangen), EB substitute dye (Beijing Prelite); Tip and Tip boxes, centrifuge tubes, glass test tubes, plates, Erlenmeyer flasks, etc. Consumables were purchased from Guangzhou Yunhui Test Instrument Company; other reagents were analytically pure conventional reagents.

[0078] LB solid medium: add agar powder with a ratio of 15g L-1 on the ba...

Embodiment 2

[0109] Example 2 Establishment of five-fold PCR detection method for five pathogenic bacteria in aquatic products

[0110] The materials and reagents used in the test are the same as in Example 1.

[0111] 1 multiplex PCR amplification

[0112] Start with the double PCR test first, and combine them in pairs. After the system test is stable, then introduce the third group of templates and primers, and the fourth group of templates and primers.

[0113] invA, hly, toxR, ipaH and vcc The composition of PCR reaction system for double PCR amplification of genes is as follows:

[0114] 2×PCR mix 12.5μL

[0115] Upstream primer (10μmol / L) 0.4μL, total (2~4μL)

[0116] Downstream primer (10μmol / L) 0.4μL, total (2~4μL)

[0117] (Note: Primer invA-up, down with hly - up, down Add 1 μL each, primers toxR - up, down , iP - up, down with vcc - up, down Add 0.4μL each)

[0118] DNA template 1.0 μL

[0119] 10 templates for the experimental bacteria Salmonella...

Embodiment 3

[0146] Five kinds of pathogenic bacteria in the aquatic product of embodiment 3 quintuple PCR detection method inspection artificial infection sample

[0147] The materials and reagents used in the test are the same as in Example 1; the five-fold detection reaction system and thermal cycle parameters are the same as in Example 2.

[0148] In order to verify the validity of the five-fold PCR detection method, 21 samples of Salmonella typhimurium, Salmonella paratyphi A, Listeria monocytogenes, Vibrio parahaemolyticus, Shigella flexneri, Fresh samples infected with non-O1 Vibrio cholerae. Infected samples were cultured with shaking at 37°C for 24 hours.

[0149] The results show that the positive detection rate of the samples by the strain isolation method in Table 3 is 100% (21 / 21), and the positive detection rate of the samples by the five-fold PCR detection method is also 100% (21 / 21). Totally Suitable.

[0150] Table 3 Comparison of the results of samples infected by fi...

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Abstract

The invention discloses primers and method for simultaneously detecting various pathogenic bacteria. The nucleotide sequences of the primers are shown in the specification, wherein an invA primer pair is used for detecting salmonella infection and salmonella paratyphi, a hly primer pair is used for detecting Listeria monocytogenes, a toxR primer pair is used for detecting vibrio parahaemolyticus, an ipaH primer pair is used for detecting shigella flexneri, and a vcc primer pair is used for detecting non-O1 Vibrio cholerae. The invention also discloses a multiple PCR (Polymerase Chain Reaction) amplification system built by using all the primers pairs, the system can be used for quickly and accurately detecting whether the corresponding pathogenic bacteria exist in a sample to be detected or not, and the whole detection process costs less than 2 hours, so that the system is simple and convenient to use; as the specificity of each primer pair is very high, a false positive result is avoided; and the primers can be made into a kit and the like, thus being very suitable for the pathogenic bacteria detection of agricultural and sideline products.

Description

technical field [0001] The invention relates to a safety detection technology for agricultural by-products, in particular to primers and a detection method for simultaneously detecting multiple pathogenic bacteria. Background technique [0002] Food safety is the most important part of public health issues. According to the report of the World Health Organization, there are as many as 100 million cases of food-borne diseases in the world every year, and about 1.7 million children worldwide die from diarrhea caused by food-borne microbial contamination every year. disease, with billions of infections among adults. The study found that the following problems exist in the field of food safety in my country: (1) Microbial contamination is the most important factor affecting food safety in my country. (2) There are still potential safety hazards in the supply of inputs, the environment of agricultural production areas, the epidemic prevention system, and the production, processi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/42C12R1/63C12R1/01
Inventor 谢青梅吕英姿张凌俊孙宝丽毕英佐
Owner 广州金苗动保科技有限公司
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