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Detection kit and detection method for brucellae in meat products

A detection method and technology for Brucella are applied to the composition and kit used for detection, and the detection and typing of 6 species of Brucella in meat products by PCR-DHPLC can solve the problems of easy contamination, false Positive, time-consuming and other problems, to achieve the effect of reduced detection cost, strong specificity, and high sensitivity

Inactive Publication Date: 2012-01-11
曹际娟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Existing detection technologies such as bacterial culture and biochemical identification, immunological technology, PCR technology, etc. have some problems: conventional biochemical identification methods are complicated to operate and take a long time, and often only one or a few types of bacteria can be identified in one detection experiment; Although immunological technology is highly sensitive, it is prone to contamination and often has false positives; PCR technology is generally used in combination with gel electrophoresis technology, and the results of electrophoresis cannot be used as the final conclusion, and other probe hybridization experiments are required
Moreover, during the isolation and identification process of Brucella, it is easy to cause laboratory pollution, and it is the pathogenic bacteria of severe infectious diseases that are prone to laboratory infection.

Method used

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  • Detection kit and detection method for brucellae in meat products
  • Detection kit and detection method for brucellae in meat products
  • Detection kit and detection method for brucellae in meat products

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1, the establishment of Brucella detection kit and detection method in meat products

[0061] (1) Establishment of detection kit for Brucella in meat products

[0062] The inventor compares and analyzes 6 kinds of Brucella homologous sequences, and designs the universal primer pair of Brucella:

[0063] Upstream primer: 5'-TGGCTCGGTTGCCAATATTCAA-3'

[0064] Downstream primer: 5'-CGCGCTTGCCTTTCAGGTCTG-3'

[0065] On this basis, assemble a test kit for Brucella detection in meat products, which includes Taq DNA polymerase and PCR reaction solution with a concentration of 5U / μL; the PCR reaction solution contains 10mM Tris HCl, 50mM KCl, 25mM MgCl 2 , dNTP (dATP, dGTP, dCTP and dTTP) each 2.5mM and the above-mentioned Brucella universal detection primer 0.2mM.

[0066] (2) Establishment of the detection and classification detection method for Brucella in meat products:

[0067] ①Preparation of the sample to be tested: the DNA genome of the sample to be tested ...

Embodiment 2

[0100] Embodiment 2, specificity test

[0101]The DNA template library of reference strains listed in Table 1 was taken, and tested according to the method established in the examples. The test results showed that only 6 kinds of Brucella had positive absorption peaks, and the rest of the strains had no positive absorption peaks, showing high detection specificity for Brucella.

[0102] In order to confirm that this positive absorption peak is the fragment of Brucella specific gene, the present invention carries out cloning and sequencing after the positive absorption peak product of DHPLC is recovered, and the result is compared with the DNA sequence of Brucella OMP, has proved DHPLC positive The absorption peak is a fragment of OMP gene of Brucella.

[0103] The test results show that the universal PCR detection primers for Brucella can be amplified by PCR, and can specifically detect 6 kinds of Brucella of the genus Brucella under the non-denaturing analysis condition of D...

Embodiment 3

[0104] Embodiment 3, sensitivity test

[0105] According to the method for enriching bacteria established in Example 1, use the turbidimetric method to quantify the bacterial solution concentration of the compound enriched bacteria cultivated, use the method established in Example 1 to extract DNA and detect, the test results are as attached figure 2 As shown, the DHPLC peak shapes from high to low are: 2.7×10 4 CFU / mL cfu / mL Brucella; 2.2×10 3 cfu / mL Brucella; 210cfu / mL Brucella; 19cfu / mL Brucella. The results show that the detection limit of the method of the present invention can reach about 0.95pg / ul Brucella DNA, and the lower detection limit can detect about 19 Brucella.

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Abstract

The invention discloses a detection kit and a detection method for brucellae in meat products. The kit comprises Taq DNA polymerase with a concentration of 5U / mu L and a PCR reaction solution, wherein the PCR reaction solution contains 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 2.5 millimols of dNTP and 0.2 millimol of general brucella detection primer. The kit and the detection method of the invention can detect 6 species of brucellae of genus brucella and detect the 6 species of brucellae respectively. The kit has high specificity and sensitivity and can detect brucellae with a concentration of 19 CFU / mL. The detection method has the advantages of quick, simple and convenient operation and low detection cost.

Description

technical field [0001] The invention relates to a detection and identification method for the genus Brucella in meat products, including Brucella sheep, Brucella bovis, Brucella pig, Brucella sheep, Brucella dog, forest The invention relates to a simultaneous detection method for six kinds of Brucella of murine bacteria, in particular to a method for detecting and typing six kinds of Brucella in meat products by using PCR-DHPLC. It also relates to the composition used for the detection, ie the kit. Background technique [0002] Existing detection technologies such as bacterial culture and biochemical identification, immunological technology, PCR technology, etc. have some problems: conventional biochemical identification methods are complicated to operate and take a long time, and often only one or a few types of bacteria can be identified in one detection experiment; Although immunological technology is highly sensitive, it is prone to contamination and often has false pos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N30/02C12R1/01
Inventor 曹际娟郑秋月王硕
Owner 曹际娟
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