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Core SNP (Single Nucleotide Polymorphism) marker for distinguishing brucella melitensis and brucellaa bortus and application of core SNP marker

A technology for Brucella bovis and Brucella melis, applied in the field of molecular biology, can solve the problems of complex operation and long time required, and achieve efficient identification

Active Publication Date: 2017-05-24
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional identification of Brucella species and biotypes is based on the growth of CO 2 The demand test, hydrogen sulfide production test, phage lysis test, thionine and basic fuchsin dye bacteriostatic test and single specific serum (A, M and R serum) agglutination test, this biotyping method is the gold standard, The disadvantage is that the operation is complicated and takes a long time

Method used

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  • Core SNP (Single Nucleotide Polymorphism) marker for distinguishing brucella melitensis and brucellaa bortus and application of core SNP marker
  • Core SNP (Single Nucleotide Polymorphism) marker for distinguishing brucella melitensis and brucellaa bortus and application of core SNP marker
  • Core SNP (Single Nucleotide Polymorphism) marker for distinguishing brucella melitensis and brucellaa bortus and application of core SNP marker

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1 is used to distinguish the acquisition of the core SNP marker of sheep species and brucella bovis

[0027] 1. Brucella strain selection

[0028] The research subjects selected 20 strains of Brucella preserved in the Brucella strain collection of the Institute of Brucella Infectious Diseases Prevention and Control, Chinese Center for Disease Control and Prevention. Among them, there are 15 sheep breeds, 3 cattle breeds and 2 pig breeds. The strains were collected and isolated from 10 provinces (autonomous regions) including Inner Mongolia Autonomous Region, Heilongjiang Province, Shandong Province, and Gansu Province, and verified by morphological and biochemical identification to determine the type of the strain. The other 55 bacteria (including one reference strain) in this example were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database (http: / / www.ncbi.nlm.nih.gov / genome / ).

[0029] 2. Genomic DNA extraction of Bru...

Embodiment 2

[0065] Example 2 Two pairs of primer detection sensitivity and specificity evaluation

[0066] 1. Sensitivity evaluation of two pairs of primers

[0067] The nucleic acids of the standard strains of Brucella melis 16M and Brucella bovis 544A were respectively diluted to a concentration of 1:1, 1:10, 1:100, 1:1000, 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 . Two pairs of primers were used to amplify, and the amplification results were shown in figure 1 (Upper is BOV_A0392 primer, lower is BOV_0551 primer). The results showed that the sensitivity of BOV_0551 primer was higher than that of BOV_A0392, and it was preferentially used as a primer for identification (Table 3).

[0068] table 3

[0069]

[0070] 2. Evaluation of detection specificity of two pairs of primers

[0071] Bartonella henselae, Vibrio cholerae, Francisella tularensis, Escherichia coli O:157, Escherichia coli O:16, Enterocolitica yeres that have serological cross-reactivity with Brucella or are clo...

Embodiment 3

[0072] Application of two pairs of primers in the detection of local brucella bacterial strains in embodiment 3

[0073] PCR was performed on the DNA of 100 strains of Brucella (60 strains of Goreum, 50 strains of Bovis, 30 strains of Suis and 20 strains of Canis) isolated in different ages and regions and had been biochemically identified Post-amplification sequencing evaluation. Specific SNP information appeared in all M. melis and B. bovis, with a specificity of 100%. For the sequence comparison results of the amplified products of some strains to be tested, see figure 2 .

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Abstract

The invention provides a core SNP ((Single Nucleotide Polymorphism)) marker for distinguishing brucella melitensis and brucellaa bortus and application of the core SNP marker. The core SNP marker for distinguishing the brucella melitensis and the brucellaa bortus is obtained via screening specific SNP sites in each MCG (Minimum Core Genome) Group based on an MCG parting technology; the core SNP markers are respectively located on brucella genes BOV_0551 and BOV_A0392, and a primer pair is designed to separate strains so as to realize simple and efficient distinguishing; the core SNP marker can be applied to brucella pathogen monitoring and epidemiological analysis, and a novel technical support is supplied to brucella prevention and control.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a core SNP marker for distinguishing brucella of ovine species and bovine species and its application. Background technique [0002] Brucella is an intracellular parasitic Gram-negative coccus that causes brucellosis (referred to as brucellosis). Brucellosis is an important zoonotic disease worldwide, causing huge economic losses to endemic regions and countries and seriously endangering human health. Before 2006, Brucella was divided into 6 species and 19 biotypes in total, including 8 biotypes of Brucella abortus, 3 biotypes of Brucella melitensis, and 5 biotypes of Brucella suis. 1 biotype, 1 dog breed (Brucella canis), 1 sheep epididymis species (Brucella ovis) and 1 desert forest wild mouse species (Brucellaneotomae) each. At present, 5 new species of Brucella have been added: Brucella ceti (isolated from whales and dolphins), Brucella pinnipedialis (isolated from pinniped...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q2600/156
Inventor 姜海张雯赵娜崔步云朴东日赵鸿雁田国忠狄栋栋范伟兴
Owner ICDC CHINA CDC
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