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Detection kit and detection method for 8 species of pathogenic bacteria in dairy products

A detection kit and detection method technology are applied to the detection kit for 8 pathogenic bacteria in dairy products and the detection field thereof, which can solve the problems of slow growth, low detection rate, and high requirements on culture conditions, and save labor. and financial resources, the detection time is short, and the operation is simple.

Inactive Publication Date: 2009-11-04
曹际娟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a lack of stable and reliable identification methods for mixed microbial samples, uncommon, harsh, and mutated bacteria
Pathogenic Aeromonas hydrophila, etc. have high requirements on culture conditions, slow growth, long detection and identification cycle, and the detection rate of traditional culture and biochemical detection methods is not high

Method used

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  • Detection kit and detection method for 8 species of pathogenic bacteria in dairy products
  • Detection kit and detection method for 8 species of pathogenic bacteria in dairy products
  • Detection kit and detection method for 8 species of pathogenic bacteria in dairy products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the establishment of 8 kinds of pathogenic bacteria detection kits and detection methods in dairy products

[0049] (1) Design and synthesis of primers and assembly of kits:

[0050]

[0051]

[0052] On this basis, a kit for the detection of 8 kinds of pathogenic bacteria in dairy products was designed, which included two sets of detection solvents: detection solution A and detection solution B;

[0053] Among them, detection solution A contains 10mM Tris·Cl, 50mM KCl, 25mM MgCl 2 , dNTP mixture (including 2.5 mM each of dATP, dGTP, dCTP and dTTP), 5 U / μL of Taq DNA polymerase, and 10 μM each of primer pairs of Enterobacter sakazakii, Serratia marcescens, Pseudomonas aeruginosa and Proteus vulgaris;

[0054] Detection solution B contains 10mM Tris Cl, 50mM KCl, 25mM MgCl 2 , dNTP mixture (including dATP, dGTP, dCTP and dTTP each 2.5mM), Taq DNA polymerase 5U / μL and pathogenic Aeromonas hydrophila, Proteus mirabilis, Klebsiella pneumoniae and Serra...

Embodiment 2

[0078] Embodiment 2, comparison with conventional PCR-electrophoresis detection method

[0079] The detection kit and detection method established in Example 1 involve two groups of PCR systems. The first group of composite PCR systems detects Enterobacter sakazakii, Serratia marcescens, Pseudomonas aeruginosa and Proteus vulgaris at one time; The PCR system detects pathogenic Aeromonas hydrophila, Proteus mirabilis, Klebsiella pneumoniae and Serratia odorans at one time.

[0080] According to the method established in Example 1, the DNA of the standard bacterial strain was extracted as a template, and amplified according to the PCR conditions of the method established in Example 1: the first group of Enterobacter sakazakii, Serratia marcescens, Pseudomonas aeruginosa and The expected amplified fragments of the four kinds of Proteus vulgaris are: 228bp, 332bp, 396bp, 413bp; the second group of pathogenic Aeromonas hydrophila, Proteus mirabilis, Klebsiella pneumoniae and Serrat...

Embodiment 3

[0083] Embodiment 3, PCR-DHPLC detection method specificity test

[0084] Take the test strains listed in Table 1, extract the genomic DNA after culture, and establish the template library. Then, using these genomic DNAs as templates, the method established in Example 1 was used for PCR-DHPLC analysis and detection.

[0085] Table 1

[0086]

[0087]

[0088]

[0089]The results of the specificity test show that the detection kit and detection method established in Example 1 can specifically detect 8 kinds of target pathogenic bacteria after two detections; Serratia aeruginosa, Pseudomonas aeruginosa and Proteus vulgaris detected specific sample peaks; detection using detection reagent B, only pathogenic Aeromonas hydrophila, Proteus mirabilis, Klebsiella pneumoniae and odor Serratia detected a specific sample peak, and the detection results of other control bacteria were all negative, without false positive and false negative results, and had high specificity.

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Abstract

The invention discloses a detection kit and a detection method for 8 species of pathogenic bacteria in dairy products, which are used for food microbial contamination monitoring and detection. The kit comprises a detection solution A and a detection solution B, wherein the detection solution A and the detection solution B both contain 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 2.5 millimols of dNTP and Taq DNA polymerase with a concentration of 5U / mu L and contain specific primer pairs of respective 4 species of target bacteria in an amount of 10 mu M each. The detection kit and the detection method of the invention can detect the 8 species of pathogenic bacteria in the diary products with high sensitivity, is short in detection time, simple and quick in operation, and can save a large amount of labor and financial resources and meet requirements for quick detection.

Description

technical field [0001] The invention relates to a detection kit for eight kinds of pathogenic bacteria in dairy products and a detection method thereof, in particular to a detection method using mPCR-DHPLC technology for detection and used reagents. . Background technique [0002] In 2004, after the exposure of the "big head doll" fed by inferior milk powder in Fuyang, Anhui Province, the safety of milk powder has become the focus of the whole society. All eyes are focused on the food safety of milk powder and so on. A survey was conducted on the contamination of infants with harmful pathogenic bacteria in different brands of milk powder at home and abroad and imported raw milk powder. It was found that the milk powder was contaminated with Enterobacter sakazakii, Klebsiella Pnaumoniae, Proteuse, and Grape Coccus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus, Serratia macescens, Serratia odorifera, etc. have a high probability of contamination. Milk powder is a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02C12R1/425C12R1/22C12R1/37C12R1/385C12R1/01
Inventor 杨春华
Owner 曹际娟
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