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Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method

A multiplex fluorescence quantitative and fluorescent quantitative technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., to achieve high sensitivity, good specificity, and strong specificity.

Active Publication Date: 2012-09-19
广州金苗动保科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There is currently no set of detection methods for the presence of the above-mentioned pathogenic bacteria that can be simultaneously detected by fluorescent quantitative PCR technology

Method used

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  • Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method
  • Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method
  • Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Design and Screening of Five Kinds of Pathogenic Bacteria Detection Primers

[0072] 1.1 Experimental materials

[0073] ABI7500 fluorescent quantitative PCR instrument was used in this experiment. Main tools Enzymes, biochemical reagents and kits:

[0074] LB broth, various biochemical tubes, Listeri enrichment solution and other reagents were purchased from Guangzhou Huankai Biological Reagent Co., Ltd.

[0075] Bacterial Genomic DNA Extraction Kit (Tiangen), DNA Recovery Kit (Tiangen), and pGM-T19 Ligation Kit (including vector, ligase) were purchased from Guangzhou Ruizhen Biotechnology Co., Ltd., and a small amount of plasmid DNA was extracted Reagent kit (Tiangen), consumables such as centrifuge tubes, glass test tubes, plates, and triangular flasks were purchased from Guangzhou Yunhui Testing Instrument Company; other reagents were analytically pure conventional reagents.

[0076] LB solid medium: add agar powder at a ratio of 15g / L on the basis of...

Embodiment 2

[0101] Example 2 Establishment of Quadruple Fluorescent Quantitative PCR Detection Method for Five Kinds of Pathogenic Bacteria in Aquatic Products

[0102] The materials and reagents used in the test are the same as in Example 1.

[0103] 1 Multiplex fluorescent quantitative PCR amplification

[0104] 1.1 invA, hly, ipaH with vcc Gene Multiplex Fluorescent Quantitative PCR Method

[0105] Start with the double fluorescent quantitative PCR test, and combine them in pairs. After the system test is stable, then introduce the template, primers and probes of the third group, and the templates, primers and probes of the fourth group.

[0106] invA, hly, ipaH with vcc The composition of PCR reaction system for double fluorescent quantitative PCR amplification of genes is as follows:

[0107]

[0108] Templates for the experimental bacteria Salmonella typhimurium, Listeria monocytogenes, Shigella flexneri, Vibrio cholerae non-O1 group 10 7 Genomic DNA extracted by...

Embodiment 3

[0133] Example 3 Five kinds of pathogenic bacteria in aquatic products quadruple fluorescent quantitative PCR detection method to test artificially infected samples

[0134] The materials and reagents used in the test are the same as in Example 1; the detection reaction system and thermal cycle parameters of the quadruple fluorescence quantitative PCR are the same as in Example 2.

[0135] In order to verify the effectiveness of the quadruple fluorescent quantitative PCR detection method, the present embodiment detects 22 samples of Salmonella typhimurium, Listeria monocytogenes, Vibrio parahaemolyticus, Shigella flexneri, and non-O1 group respectively. Vibrio cholerae infects fresh samples. Infected samples were cultured with shaking at 37°C for 24 hours.

[0136] The results show that: the positive detection rate of samples by strain isolation method in Table 3 is 100% (22 / 22), and the positive detection rate of samples by quadruple fluorescence quantitative PCR detection...

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Abstract

The invention discloses fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and a detection method, and belongs to the field of molecular detection. Five groups of the fluorescent quantitative PCR primers and the probes are provided and can be used for detecting salmonella, listeria monocytogenes, vibrio parahaemolyticus, shigella flexneri and vibrio cholerae non-O1 respectively, wherein except the primers and the probes for listeria monocytogenes genes and the primers and the probes for vibrio parahaemolyticus genes cannot be used simultaneously, the pathogenic bacteria which can be detected by the corresponding primers and probes can be detected simultaneously by combining any other two groups or more than two groups of the primers and probes. The invention also discloses multiple fluorescent quantitative PCR amplification systems which are constructed by using the primers and the probes, so the primers and the probes can detect whether samples to be detected contain the corresponding pathogenic bacteria or not quickly and accurately, are convenient to operate and high in specificity, can be made into kits and the like and are applicable to the detection of the pathogenic bacteria of the subsidiary agricultural products, and a false positive result is avoided.

Description

Technical field [0001] The present invention involves agricultural and sideline product safety testing technology, and specifically involves the simultaneous detection of fluorescent quantitative PCR primers and probes and detection methods of a variety of aquatic products. Background technique [0002] Food safety is the most important part of public health issues. At present, there are microbial pollution in the field of food safety in my country; there are still hidden safety hazards in the production, processing and sales of agricultural products; insufficient food safety scientific and technological achievements and technological reserves; food safety standard systems, inspection inspection There are still obvious discomfort in the testing system and certification recognition system. The food safety management system and laws and regulations are needed to be improved; a series of food safety issues such as food poisoning and food -oriented diseases. [0003] Therefore, only ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04C12R1/42C12R1/63C12R1/01
CPCY02A50/30
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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