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SNP molecular marker used for identifying Brucella vaccine strain M5, and application of SNP molecular marker

A Brucella and molecular marker technology, applied in the direction of microbial-based methods, recombinant DNA technology, microbial determination/inspection, etc., can solve problems affecting animal brucellosis monitoring and epidemic management, differential diagnosis, human infection, etc. Achieve the effect of simple and efficient identification, rapid detection and typing, and efficient technical support

Active Publication Date: 2021-10-08
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Brucella vaccine strain M5 may cause human infection, and the antibodies produced by animals inoculated with M5 vaccine are difficult to differentiate from antibodies produced by naturally infected wild strains, which affects animal brucellosis surveillance and outbreak management

Method used

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  • SNP molecular marker used for identifying Brucella vaccine strain M5, and application of SNP molecular marker
  • SNP molecular marker used for identifying Brucella vaccine strain M5, and application of SNP molecular marker
  • SNP molecular marker used for identifying Brucella vaccine strain M5, and application of SNP molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 is used to identify the acquisition of the core SNP molecular marker of Brucella vaccine strain M5

[0059] 1. Systematic analysis of the whole genome information of Brucella using the minimal core component typing method

[0060] (1) Download Brucella melis (136 strains in total) from the GenBank database (http: / / www.ncbi.nlm.nih.gov / genome / ) of the National Center for Biotechnology Information (NCBI) The Complete and Scaffold genomes.

[0061] (2) Based on the Blast method, the gene of M5GCA_000348645.1 was compared with the genomes of 135 strains of Brucella in sequence. If the similarity is greater than 60% and the compared sequence exceeds 70% of the original sequence, the gene is considered to exist in this in the genome. According to this analysis strategy, a total of 1291 Core genes were found in all strains.

[0062] (3) Using the Mummer program, the Core gene sequence was sequentially compared with 134 genomes, the SNP sites were found, and the...

Embodiment 2

[0079] Example 2 Specificity Evaluation of SNP Molecular Marker Detection

[0080]Bartonella henselae, Vibrio cholerae, Francisella tularensis, Escherichia coli O:157, Escherichia coli O:16, Enterocolitica with serological cross-reactivity with Brucella or close relatives to Brucella species Mori bacteria O: 9, Staphylococcus aureus and other kinds of Brucella wild strains, multiple vaccine strains such as S2 (pig species), A19 (cattle species) etc. are used as specific controls as samples to be tested, for example 1 The screened SNP molecular markers and their primer pairs and probe pairs (see Table 1) were evaluated for detection specificity. The specific methods are as follows:

[0081] 1. Genomic DNA extraction

[0082] Bacterial genomic DNA extraction kits from Beijing Tiangen Biochemical Reagent Co., Ltd. were used in strict accordance with the instructions in the kits, including the above-mentioned Bartonella henselae, Vibrio cholerae, Francisella tularensis Escherichi...

Embodiment 3

[0089] The application of embodiment 3 SNP molecular marker in distinguishing Brucella vaccine strain M5 and place Brucella bacterial strain

[0090] Utilize the SNP molecular marker that screening obtains in embodiment 1 and its primer (SEQ ID NO.2-3) and probe (SEQ ID NO.4-5) to Brucella vaccine strain M5, Brucella standard strain, 120 Brucella wild strain isolated from local strains and Brucella vaccine strain S2 (pig species), A19 (cattle species) are differentiated and identified, and the extraction of genomic DNA and the RT-PCR method are the same as in Example 2.

[0091] The identification results are shown in Table 2, Table 3 and Table 4. The fluorescence signal of the amplification curve of Brucella vaccine strain M5 is the fluorescence (FAM) labeled with the probe shown in SEQ ID NO.4, and the corresponding SNP site is A, The fluorescence signal of the amplification curve of each standard strain (see Table 4 for the name of the standard strain) is the fluorescence (...

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Abstract

The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker used for identifying a Brucella vaccine strain M5, and application of the SNP molecular marker. The SNP molecular marker provided by the invention comprises a nucleotide sequence of which the polymorphism is A / C at the 37th site of a sequence as shown in SEQ ID NO.1, the polymorphism site of the SNP molecular marker is A and corresponds to the Brucella vaccine strain M5, and the polymorphism site of the SNP molecular marker is C and corresponds to a wild strain or other Brucella vaccine strains. The SNP molecular marker and a detection primer and a probe thereof can realize simple, convenient and efficient identification of the Brucella vaccine strain M5, the wild strain and other Brucella vaccine strains, are relatively high in accuracy, can be applied to Brucella disease pathogen monitoring and epidemiological analysis, and provide accurate technical support for Brucella disease prevention and control.

Description

technical field [0001] The invention relates to the technical field of molecular markers, in particular to SNP molecular markers for identifying Brucella vaccine strain M5 and applications thereof. Background technique [0002] Brucella is an intracellular parasitic Gram-negative coccus that causes brucellosis (referred to as brucellosis). 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 each of biotype, dog breed (Brucella canis), sheep epididymis species (Brucella ovis) and desert forest voles species (Brucella neotomae). At present, 5 new species of Brucella have been added: Brucella ceti (isolated from whales and dolphins), Brucella pinnipedialis (isolated from pinnipeds), Brucella microti (isolated from voles), Brucellainopinata (isolated from blood and wound exudate) and Brucella papionis (isolated from baboon). The G+C con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 姜海张雯朴东日杨晓雯田国忠赵鸿雁范玉
Owner ICDC CHINA CDC
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