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Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product

A technology of multiple fluorescent quantitative and detection methods, applied in the direction of microorganism-based methods, microorganism measurement/inspection, fluorescence/phosphorescence, etc., to achieve the effect of promoting food export and improving food hygiene quality

Active Publication Date: 2012-11-07
中华人民共和国舟山出入境检验检疫局
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Problems solved by technology

[0004] Although a lot of research has been done in the prior art, in the same reaction tube, a fluorescent PCR detector with multiple laser channels is used to establish a multiplex detection system for simultaneous detection of Vibrio parahaemolyticus, Vibrio cholerae and Listeria monocytogenes. Fluorescent quantitative PCR reaction system has not been reported and related literature is published

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  • Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product
  • Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product
  • Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product

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Embodiment

[0042] Implementation example: the detection of fresh shrimps (A, B two samples), specifically according to the following steps:

[0043] 1. Take 25g shrimp samples, cut them into pieces, mix them with 225mL enrichment solution, and incubate at 37±1°C for 4-6h;

[0044] 2. Take 1.5mL enrichment solution into a centrifuge tube, centrifuge at 12000rpm for 3min, and discard the supernatant;

[0045] 3. Resuspend the pellet with 1 mL of ddH2O, centrifuge at 12,000 rpm for 2 min, discard the supernatant, and repeat the operation once;

[0046] 4. Boiling method to extract the genomic DNA of samples A and B;

[0047] 5. Take 18 μL of the PCR reaction mixture and add it to the PCR reaction tube, then add 2 μL of DNA respectively;

[0048] 6. After setting according to the above procedure, put the PCR reaction tube into the fluorescent quantitative PCR instrument for detection;

[0049] 7. Analyze the specific S curve and the Ct value of the sample to be tested, and judge the resul...

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Abstract

The invention discloses a multiplex fluorescent quantitative polymerase chain reaction (PCR) detection method for main pathogenic bacteria in an aquatic product. The method is characterized by comprising the following steps of: (1) performing enrichment culture on a sample to be detected in enrichment solution for 4 to 8 hours; (2) extracting genome DNA from the cultured sample by adopting a boiling method; (3) performing multiplex fluorescent quantitative PCR detection by using specific primers and TaqMan probes; and (4) performing result judgment according to the detected specific S amplification curve and threshold value (Ct value). Compared with the prior art, the method has the advantages that: one-tube multi-detection actual requirement can be met, and a solid technical guarantee can be provided for quick, sensitive and specific detection of vibrio parahaemolyticus, vibrio cholerae and listeria monocytogenes.

Description

technical field [0001] The invention relates to a multiple fluorescence quantitative PCR detection method for Vibrio parahaemolyticus, Vibrio cholerae and Listeria monocytogenes in aquatic products. Background technique [0002] In recent years, food-borne diseases have shown an upward trend. These diseases are mainly caused by food-borne pathogens, including Vibrio parahaemolyticus, Listeria monocytogenes and food-borne pathogens that exist in aquatic animals or farming environments for a long time. Vibrio cholerae, etc. However, due to the small amount of bacteria, the large variation of strains and the limitations of detection methods, traditional detection methods are often unable to detect or easily miss detection. Therefore, it is of great significance to seek rapid, sensitive and specific methods to detect Vibrio parahaemolyticus, Vibrio cholerae and Listeria monocytogenes in aquatic products. [0003] The detection technology of foodborne pathogenic bacteria has un...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/63C12R1/01
CPCY02A50/30
Inventor 周向阳王淑娜沈飚胡兴娟贝文联朱应伟徐君辉周秀锦邵宏宏
Owner 中华人民共和国舟山出入境检验检疫局
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