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Preprocessing method for detecting salmonella live bacteria DNA detection in meat and meat products

A technology for Salmonella and meat products, applied in the field of Salmonella detection, can solve the problems of food safety threats, reducing the effectiveness of pathogenic bacteria detection, unable to identify live bacteria and dead bacteria, etc.

Inactive Publication Date: 2014-03-26
哈尔滨海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Genetic testing is one of the rapid detection methods for pathogenic bacteria, but it is currently unable to identify live and dead bacteria, which reduces the effectiveness of pathogenic bacteria detection
Because only "live" bacteria can retain the virulence and pathogenicity of the original bacteria, it poses a potential threat to food safety

Method used

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  • Preprocessing method for detecting salmonella live bacteria DNA detection in meat and meat products
  • Preprocessing method for detecting salmonella live bacteria DNA detection in meat and meat products

Examples

Experimental program
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Effect test

Embodiment 1

[0024] A method based on quantitative PCR method for the detection of live Salmonella in simulated chilled pork samples

[0025] 1. Materials and reagents

[0026] Experimental strain: Salmonella (CMCC(B)50115).

[0027] Food: Chilled pork, bought in the market.

[0028] Main reagents:

[0029] DNA extraction solution formula: 1M Tris-HCl 100 mL; 5M NaCl 20 mL; 0.5M EDTA 100 mL; 20% SDS 100 Ml; adjust pH to 8.0; ddH2O to 4 L.

[0030] PMA: Dissolve 1 mg of PMA in 200 μL of 20% DMSO solution to obtain a stock solution of 5 μg / μL, and store it at -20°C in the dark. When in use, dilute the PMA stock solution 10 times to obtain the PMA working solution.

[0031] 2. Bacterial culture and preparation of heat-killed bacteria

[0032] (1) Bacterial culture - preparation of live bacteria

[0033] The Salmonella strains were activated, streaked on HE agar medium, and cultured in a constant temperature incubator at 37°C for 24h. Pick a single colony and inoculate it in 50 mL of nu...

Embodiment 2

[0042] Fluorescent quantitative PCR detection

[0043] 1. Design of primers and probes

[0044] Fluorescent PCR was performed using the extracted sample genomic DNA as a template to qualitatively detect the target bacteria in the sample. The primer sequences used to design fluorescent quantitative PCR are as follows:

[0045] The upstream primer Pf is: 5'-TCGTCATTCCATTACCTAC-3'

[0046] The downstream primer Pr is: 5'-AAAC GTTGAAAAACTGAGGAC-3'

[0047] The probe Pb is: 5'-FAM-TCTGGTTG ATTTCCTGATCGCA-BHQ1.

[0048] 2. Fluorescent quantitative PCR detection

[0049] ABI 7500 fluorescent PCR instrument was used to detect 2 samples, and the mixture was pre-denatured at 95°C for 2 minutes; then 95°C for 10 s; 60°C for 40 s (collecting fluorescence signals), a total of 40 cycles; and finally incubated at 40°C for 10 minutes. Test results such as figure 1 shown.

[0050]

[0051] Described mixed solution is composed as follows:

[0052] 10×(NH4) 2 SO 4 buffer ...

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Abstract

The invention discloses a preprocessing method for detecting salmonella live bacteria DNA in meat and meat products. The method comprises the following steps: firstly processing meat and meat products, adding PMA into a processed sample, controlling the final concentration of the PMA to be 15 micrograms / mL, sufficiently and uniformly mixing the mixture, and incubating for 3min in a dark place; illuminating for 3min at the position of 20cm below a 500w halogen tungsten lamp; extracting the genome DNA of the processed sample; and performing real-time fluorescence PCR quantitative detection on salmonella live bacteria in the sample by taking the extracted genome DNA as a template. The method can rapidly detect pathogenic live bacteria in meat and meat product matrix, so that the interference of food matrix turbidity and bacterial pollution background values is avoided, and the detection effectiveness is improved.

Description

technical field [0001] The invention relates to a method for detecting salmonella, in particular to a method for detecting live salmonella in meat and meat products. Background technique [0002] Genetic testing is one of the rapid detection methods for pathogenic bacteria, but it is currently unable to identify live and dead bacteria, which reduces the effectiveness of pathogenic bacteria detection. Because only "live" bacteria can retain the virulence and pathogenicity of the original bacteria, they pose a potential threat to food safety. The "dead" bacteria that have been inactivated by various means such as high temperature and ultraviolet rays have lost the virulence and pathogenicity of the original bacteria, and will not threaten food safety. Therefore, the key to the detection of pathogenic bacteria is to distinguish between "live" bacteria and "dead" bacteria. Research on detection methods to distinguish live bacteria from dead bacteria is a key link and technolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/6806C12Q2523/313C12Q2563/173
Inventor 李苏龙徐义刚鞠文东程成刘忠梅肖性龙
Owner 哈尔滨海关技术中心
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