PCR method for quickly detecting brucella in milk sample

A Brucella and milk technology, applied in the field of biological detection, can solve the problems of backward quarantine diagnosis, etc., and achieve the effect of simple steps, good specificity and strong practicability

Inactive Publication Date: 2010-03-03
JILIN UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Domestic quarantine and diagnosis of brucellosis is still relatively backward, and many domestic laboratories are stil

Method used

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  • PCR method for quickly detecting brucella in milk sample
  • PCR method for quickly detecting brucella in milk sample
  • PCR method for quickly detecting brucella in milk sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Kit composition and preparation

[0040] 1) DNA extraction solution: including the following components: 10×TE (0.1M Tris-HCl, 0.1M EDTA pH8.8), 5% NP-40 and 0.5% Tween-20.

[0041] 2) PCR reaction solution: prepared reaction solution, 10×PCR Buffer (containing Mg 2+ ) 10 μL, dNTPs (2.5 mmol / L) 5 μL, forward primer and reverse primer 2 μL (10 μmol / L), Taq enzyme (5U / μL) 0.5 μL, sterile double distilled water 25.5 μL;

[0042] 3) Standard positive template: the standard positive template is the pMD-18 recombinant plasmid composed of a 287-base nucleotide fragment of the highly conserved gene BCSP31 gene of Brucella.

[0043] 4) Brucella BCSP31 gene-specific primer sequence:

[0044] Forward primer BF: 5′-ATCCAGGAAACCCGACTATGCCA-3′;

[0045] Reverse primer BR: 5′-AACCGCAAAGCTCGCTCCCAAC-3′

[0046] 5) Negative quality control standard: The negative quality control standard is sterile double distilled water.

Embodiment 2

[0048] Kit specificity test

[0049] Validated with DNA Validation for Salmonella Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus suis, Escherichia coli, Klebsiella pneumoniae, Proteus, Pasteurella multocida, Enterocolitis Yare Mori bacteria, Bacillus cereus, Actinobacillus pleuropneumoniae serotype 1, Actinobacillus pleuropneumoniae serotype 2, Actinobacillus pleuropneumoniae serotype 3, Actinobacillus pleuropneumoniae serotype 5, Escherichia coli O157:H7 and other 15 kinds of controls, 5 μL of each positive sample was used as a template for PCR reaction, and a negative control was set at the same time.

[0050] The PCR amplification conditions were cycle conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, extension at 72°C for 30 sec, and amplification for 31 cycles.

[0051] The results showed that only Brucella bovis 544A, Brucella bovis 104M, Brucella melis 16M, and Brucella suis S2 ampl...

Embodiment 3

[0053] Sensitivity test of the kit

[0054] Take 20 μL of the genome of Brucella bovis 544A, dilute it 10 times, measure its concentration with UV-2802H ultraviolet-visible spectrophotometry juice, and then double-dilution, take the double-diluted Brucella bovis 544A genome as a template, take 5 μL Carry out PCR reaction and set up negative control at the same time.

[0055] The PCR amplification conditions were cycle conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, extension at 72°C for 30 sec, and amplification for 31 cycles.

[0056] The sensitivity of the kit was determined to be 0.16pg genome (equivalent to 11 to 16 bacteria) through the experimental results (see image 3 ).

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Abstract

The invention discloses a PCR method for quickly detecting brucella in a milk sample, relating to a gene detection technology of pathogens of zoonotic infectious diseases and being applicable to qualitative detection of brucella. The PCR method consists of the components of a standard positive template, a PCR reaction solution, a brucella BCSP31 gene specific primer and a negative quality-controlstandard sample. The invention has the advantages of quick detection speed, good specificity, high sensitiveness, simple using steps, high repeatability, and capability of replacing the traditional pathogenic detection method.

Description

technical field [0001] The invention discloses a PCR method for rapidly detecting Brucella in milk samples, which is used for detecting Brucella or Brucella DNA in milk, and also provides a kit suitable for the method. It belongs to the technical field of biological detection. Background technique [0002] Brucellosis is an important zoonotic infectious disease caused by Brucella, which is listed as a B-type animal disease by the World Organization for Animal Health (OIE), and is an infectious disease that must be inspected in international trade quarantine. , my country lists it as a second-class animal disease. Brucella includes 6 species and 20 biotypes, namely, B. melitensis biotypes 1-3, B. abortus biotypes 1-9, pig species Brucella (B.suis) biotypes 1-5, Brucella ovis epididymis (B.ovis), Brucella sarlin (B.neotomae) and Brucella canis (B. .canis), among which Brucella melis, Brucella bovis, and Brucella suis are the main pathogenic bacteria that cause abortion in hu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 韩文瑜杜涛峰雷连成孙长江杜崇涛赵伟栋张俊梅冮森林王大力李铁峰
Owner JILIN UNIV
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