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PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof

A technology of Brucella and kit, which is applied in the field of PCR kit and its preparation for simultaneous detection of four kinds of Brucella in cattle, sheep, pigs and dogs, and can solve the problem that Brucella cannot distinguish the types of infection at the same time

Inactive Publication Date: 2013-11-27
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the problem that the existing serological methods for detecting Brucella cannot distinguish the types of infection at the same time, the invention provides a PCR kit for simultaneously detecting four kinds of Brucella in cattle, sheep, pigs and dogs and its preparation method and how to use

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  • PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof
  • PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof
  • PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof

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Experimental program
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Effect test

Embodiment 1

[0114] The using method of the PCR kit that simultaneously detects four kinds of brucella of cattle, sheep, pigs and dogs of the present invention is as follows:

[0115] (1) Extract sample DNA from the samples to be tested. The samples to be tested are milk, beef enriched with liquid medium, mutton enriched with liquid medium, pork enriched with liquid medium, and pork enriched with liquid medium. fungus dog meat, etc.;

[0116](2) Using the extracted sample DNA as a template, add the sample DNA, positive quality control standard and negative quality control standard into tubes containing PCR reaction solution and DNA polymerase respectively, and proceed according to the optimized PCR reaction conditions. Amplification: 95°C for 5min, 94°C for 30sec, 62°C for 30sec, 72°C for 30sec, 30 cycles, 72°C for 10min; the effect of PCR amplification directly affects the sensitivity and specificity of detection. The primer concentration, annealing temperature, annealing time, cycle tim...

Embodiment 2

[0118] The assembly of embodiment 2PCR kit

[0119] (1) Prepare PCR reaction solution:

[0120] ①Design four pairs of primers for identifying Brucella of cattle, sheep, porcine and canine: P IS711f and P 494r ,P IS711f and P 732r ,P 591f and P 591r ,P 272f and P 272r :

[0121] forward primer P IS711f : 5′-TGCCGATCACTTAAGGGCCTTCAT-3′,

[0122] reverse primer P 494r : 5′-GACGAACGGAATTTTTCCAATCCC-3′,

[0123] reverse primer P 732r : 5′-AAATCGCGTCCTTGCTGGTCTGA-3′,

[0124] forward primer P 591f : 5′-CTGGTGGGTATCGCATTACTCGG-3′,

[0125] reverse primer P 591r : 5′-TTCAGGAAAGCCTGGCGGTACTG-3′,

[0126] forward primer P 272f : 5′-GGAACACTACGCCACCTTGT-3′,

[0127] reverse primer P 272r : 5'-GATGGAGCAAACGCTGAAG-3';

[0128] ②Preparation of PCR reaction solution: The PCR reaction solution consists of four pairs of primers for identification of bovine, sheep, porcine and canine Brucella and contains dNTPs, Mg 2+ , double distilled water (ddH 2 O) PCR buffer composit...

Embodiment 3

[0157] The repeatability test of embodiment 3PCR kit

[0158] (1) Bacterial Genomic DNA Extraction Kit was used to extract Brucella from Brucella bovis S19, Brucella melis M5, Brucella suis S2, and Brucella canis RM6 / 66 respectively DNA, for PCR amplification;

[0159] (2) Take 47.8uL of each PCR reaction solution, take 2μL each of the Brucella DNA obtained in step (1) and Brucella positive quality control standard, and set up a negative control, and add them to different PCR reaction tubes respectively. Perform PCR amplification in parallel on a PCR instrument, and the PCR reaction conditions are: 95°C for 5 min, 94°C for 30 sec, 62°C for 30 sec, 72°C for 30 sec, 30 cycles, and 72°C for 10 min.

[0160] (3) Take 5 μL of the PCR product and detect it by 1.5% agarose gel electrophoresis. Observe the amplified bands of 494bp and 591bp in Brucella bovis on the gel image analyzer, and amplify the bands of Brucella melis. The bands of 732bp and 591bp were amplified, the bands of ...

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Abstract

A PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as a preparation method and a using method thereof, and belongs to the field of gene detection of anthropozoonosis pathogens. The PCR kit comprises a PCR liquid reactant, DNA polymerase, positive quality control standard substances and negative quality control standard substances; the PCR liquid reactant comprises four pairs of primers for identifying Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis and a PCR buffer solution containing dNTPs, Mg<2+> and double distilled water. The PCR kit has high practicability, can be used for directly detecting the type of Brucella in a contaminated sample, has high detection speed, high sensitivity (10<2>cfu / ml), high specificity and good repeatability, is safe, reliable, quick, simple and convenient, can be used for qualitative detection of the type of Brucella in the sample, and can replace traditional etiological and serological methods.

Description

technical field [0001] The invention relates to the technical field of genetic detection of zoonotic infectious disease pathogens, in particular to a PCR kit for simultaneously detecting four kinds of Brucella in cattle, sheep, pigs and dogs, as well as a preparation method and a use method thereof. Background technique [0002] Brucella is a kind of intracellular parasite, which is Gram-negative cocci, and cattle, sheep, pigs, dogs, horses, deer, camels, and humans are susceptible. Brucella has a strong invasive force, mainly through the digestive tract infection, and complete skin and mucous membranes can also be invaded by Brucella, causing brucellosis. [0003] Brucellosis, also known as Mediterranean relaxation fever or wave fever, is a zoonotic infectious disease prevalent worldwide. There are about 5 to 6 million patients with brucellosis in the world, and there are about 500,000 new cases every year. The population threatened by brucellosis in my country is about 35...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 王兴龙陈思王秀然钱晶郎需龙王晓旭
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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