PMA-dual ddPCR-based quantitative detection method for vibrio parahaemolyticus

A dual detection reagent technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of inability to distinguish live bacteria from dead bacteria, false positives, etc., and achieve the elimination of parahemolytic Effects of interference from Vibrio dead bacteria, high specificity, and high detection sensitivity

Inactive Publication Date: 2021-11-12
南京市食品药品监督检验院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition to normal living bacterial cells, food samples usually carry a large number of dead cells, but their DNA can exist stably. However, PCR technology is based on nucleic acid amplification, so only PCR technology cannot distinguish live bacteria. and dead bacteria, which can easily cause "false positive" results

Method used

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  • PMA-dual ddPCR-based quantitative detection method for vibrio parahaemolyticus
  • PMA-dual ddPCR-based quantitative detection method for vibrio parahaemolyticus
  • PMA-dual ddPCR-based quantitative detection method for vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the design of primer probe

[0048] According to the conserved sequences of Vibrio parahaemolyticus-specific target VP0488 (accession number: BA000031.2) and the traditional species-specific gene thermolabile hemolysin TLH (accession number: BA000032.2) self-screened by our laboratory, high Specific primers and probes are listed in Table 1. All primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0049] Table 1 PCR detection primers and probe sequences of Vibrio parahaemolyticus

[0050]

[0051]

[0052] Note: a, upstream primer; b, downstream primer; c, probe

[0053] The present invention adopts the Taqman probe method, and its main principle is to use enzymes to combine specific probes with fluorescent reporter groups (FAM, HEX) and fluorescent quencher groups (BHQ1) that are combined with templates during chain extension. The process of cutting off the fluorescent signal is reflected by monitoring the intensity o...

Embodiment 2

[0054] Example 2: Specific verification of primer probes

[0055] 1 Materials and methods

[0056] 1.1 Strains

[0057] Vibrio: Vibrio parahaemolyticus (ATCC 33847), Vibrio parahaemolyticus (ATCC 17802), Vibrio alginolyticus (ATCC 17749), Vibrio cholerae (CICC 23794), Vibrio vulnificus (ATCC 27562);

[0058] Non-Vibrio: Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Staphylococcus aureus (CMCC (B) 260031), Listeria monocytogenes (ATCC 19115), Pseudomonas aeruginosa ( ATCC 9027), Shigella flexneri (CMCC(B)51572), Bacillus cereus (CMCC(B)63303), Enterobacter sakazakii (ATCC 29544), Campylobacter jejuni (ATCC 29428);

[0059] The above strains were all preserved by the Microbiology Laboratory of Nanjing Institute of Food and Drug Administration.

[0060] 1.2 Main reagents and instruments

[0061] Premix Ex Taq (Probe qPCR) (Dalian Bao Biological Engineering Co., Ltd.); CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad, USA).

[0062] 1.3 B...

Embodiment 3

[0072] Example 3: Double ddPCR amplification conditions and reaction system optimization

[0073] 1 Materials and methods

[0074] 1.1 Strains

[0075] Vibrio paralyticus standard strain ATCC 33847.

[0076] 1.2 Main reagents and instruments

[0077] ddPCR Supermix for Probes (No dUTP), QX200 Droplet Generation Oil, and ddPCR Droplet Reader Oil were all purchased from Bio-Rad, USA; C1000 Touch gradient PCR instrument, DG8 cartridge microdroplet generation card, QX200 Droplet Generator, PX1 PCR Plate Sealer, and QX200 Droplet Reader were all purchased from From the American Bole Company.

[0078] 1.3 Bacterial DNA extraction and double ddPCR detection

[0079] Take the boiling method to extract the bacterial genome DNA, the method is the same as in Example 2.

[0080] Double ddPCR reaction system (20 μL): ddPCR Supermix for Probes (No dUTP) 10 μL, vp0488 and tlh gene upstream and downstream primers (10 μmol / L) each 1.0 μL, probe (10 μmol / L) 0.5 μL, DNA template 4 μL, and f...

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Abstract

The invention discloses a PMA-dual ddPCR-based quantitative detection method for vibrio parahaemolyticus, and belongs to the technical field of food safety detection. According to the method, a specific primer and a probe are designed and verified by taking a vibrio parahaemolyticus self-screening specific new target VP0488 and a traditional target TLH as target fragments. Meanwhile, in order to ensure the specificity and sensitivity of the method and optimize dual ddPCR amplification conditions, a reaction system and PMA treatment conditions, a PMA-dual ddPCR method for quantitatively detecting viable vibrio parahaemolyticus is established. Results show that the sensitivity of the method is higher than that of dual real-time fluorescent quantitative PCR (qPCR), and the method has the advantages of being high in specificity and good in accuracy, and has practical application value in the aspect of food safety detection.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a quantitative detection method for vibrio parahaemolyticus based on PMA-double ddPCR. Background technique [0002] Vibrio parahemolyticus (Vp) is a kind of rod-shaped or curved Gram-negative bacteria widely distributed in aquatic products such as fish, shellfish, shrimp and crab. Drinking contaminated water and consuming raw or undercooked seafood can lead to vibriosis, especially in the elderly and children who are immunocompromised. According to statistics, there are 80,000 cases of diseases caused by vibriosis in the United States every year, of which about 45,000 are caused by Vibrio parahaemolyticus. In my country, the National Food Safety Risk Assessment Center conducted a statistical analysis of national foodborne disease outbreak surveillance data and found that microbial foodborne diseases have always been an important cause of food safety pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/173C12Q2563/159C12Q2563/107Y02A50/30
Inventor 杨军周海波刘新梅别小妹程逸宇吴海晶刘延
Owner 南京市食品药品监督检验院
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