Primer pair and probe for identifying vaccine strain S2 and wild strain of Brucella and application thereof

A technology for Brucella and vaccine strains, applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms, can solve the problem of high cost of detection and typing, and achieve the goal of being suitable for large-scale promotion and implementation, accurate detection results, and improved efficiency Effect

Inactive Publication Date: 2021-06-04
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires dual-probe detection, and the cost of detection and typing is high. At present, there is an urgent need for a lower detection cost and simpler typing detection method

Method used

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  • Primer pair and probe for identifying vaccine strain S2 and wild strain of Brucella and application thereof
  • Primer pair and probe for identifying vaccine strain S2 and wild strain of Brucella and application thereof
  • Primer pair and probe for identifying vaccine strain S2 and wild strain of Brucella and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Primer and probe design screening

[0037] After screening a large number of primers and probes designed for the Brucella S2 vaccine strain, it was found that the primer pair F, R, and probe designed according to the SNP site A290C of Brucella BSS2_I0227 (CP006961.1) The P fluorescence method can most effectively distinguish the Brucella S2 vaccine strain from the wild strain. The nucleotide sequences of the primer pair F, R and the probe P are shown below.

[0038] Primer F: 5'-GCTCGACAAGGAAATCAAG-3' (SEQ ID NO: 1);

[0039] Primer R: 5'-TCAGGTCCGTGTAAAGATC-3' (SEQ ID NO: 2),

[0040] Probe P: 5'-CCAACCATTATTCTTTCGCGCCGCAATA-3' (SEQ ID NO: 3).

[0041] The fluorescent group Texas Red was labeled at the 5' end of the probe P, and the quencher group BHQ1 was labeled at its 3' end. However, other fluorescent groups and quenching groups conventional in the art can achieve the same effect.

[0042] Test results such as figure 1 As shown, at the probe P position, there is...

Embodiment 2

[0043] Embodiment 2 The establishment of fluorescent PCR detection method and melting curve analysis method

[0044] 1) Preparation of positive control substance

[0045] according to figure 1 As a result of the comparison analysis of the probe sequences, the sequence of the probe P corresponding to the Brucella S2 vaccine strain is one group, and the other wild strains are another group. Using S2 vaccine nucleic acid and wild strain 1330S as templates, primer pair DF: 5'-TTATCCTGTCACGCCTACATCCG-3' (SEQ ID NO: 4) and DR: 5'-TTCACCACCCTCGGCAACC-3' (SEQ ID NO: 5) as primers, Premix Ex-Taq amplified nucleic acid fragments with a size of 1590bp, cloned them into PMD18T Vector, and named them pS2 and pW in sequence, and used them as positive controls for S2 vaccine strain and positive controls for Brucella wild strain, respectively.

[0046] 2) Establishment of PCR system

[0047] The two positive control substances obtained in step 1) were used as templates to carry out fluores...

Embodiment 3

[0052] Embodiment 3 specific detection

[0053] The genome nucleic acid of Brucella vaccine S2 strain, Brucella bovis 544A strain, Brucella suis 1330S strain, Brucella ovis 16M strain, and porcine Escherichia coli preserved and extracted in the laboratory were respectively selected ( E.coli), Pasteurella (Pasteurella), Streptococcus suis (S.suis), Pseudomonas aeruginosa (P.aeruginosa), Actinobacillus pleuropneumoniae (App) and other common swine pathogenic genome nucleic acids, the above nucleic acids and Water (negative control) is respectively used as PCR template, adopts PCR amplification reaction and melting curve analysis method established in embodiment 2 to analyze, and compares with the analysis result of positive standard sample pS2 and pW, melting curve peak type figure Such as image 3 shown.

[0054] Depend on image 3 It can be seen that the detection method of the present invention can only specifically amplify the positive standard samples of Brucella and for...

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Abstract

The invention discloses a primer pair and a probe for identifying a vaccine strain S2 and a wild strain of Brucella and application thereof, and relates to the technical field of bacterial detection. The invention provides a primer pair comprising an upstream primer, with the nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with the nucleotide sequence as shown in SEQ ID NO.2 and a probe with the nucleotide sequence as shown in SEQ ID NO.3. According to the invention,the vaccine strain S2 and the wild strain of the Brucella can be rapidly and accurately identified. The invention also provides a method for identifying the vaccine strain S2 and the wild strain of the Brucella by using the primer pair and the probe, and the method is high in sensitivity, good in specificity and suitable for clinical application. The invention provides a new effective tool for preventing, treating and screening brucellosis, greatly improves the efficiency of brucellosis prevention and treatment, and is suitable for large-scale popularization and implementation, with huge application potential and potential commercial value.

Description

technical field [0001] The invention relates to the technical field of bacterial detection, in particular to a primer set, a probe and an application thereof for distinguishing Brucella S2 vaccine strains from wild strains. Background technique [0002] Brucellosis (also known as Brucellosis) is a zoonotic infectious disease caused by Brucella. Brucella is a Gram-negative facultative anaerobic bacterium, which can be divided into at least 10 species according to the difference in pathogenicity and host selectivity, among which Brucella melis ( Brucella melitensis), Brucella abortus and Brucella suis were the most infective species. [0003] Brucellosis is extremely harmful to animal husbandry. Its prevalence has caused serious safety hazards in meat, milk and other products in the affected areas, greatly affecting the foreign trade of cattle, sheep, pigs and other animal products, and seriously threatening human health. At present, the epidemic prevention measures against ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2563/107C12Q2527/107C12Q2600/156
Inventor 张春红马祥刘志成沈海燕孙俊颖张建峰廖明
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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