Bacillus anthracis duplex fluorescence quantitative PCR detection kit and detection method

A detection kit and Bacillus anthracis technology, which is applied in the field of fluorescent quantitative PCR detection, can solve problems such as cross-reaction, anthrax diagnosis troubles, distinguish Bacillus anthracis from other Bacillus cereus, etc., and achieve rapid detection and broad application prospects Effect

Inactive Publication Date: 2019-01-18
王素华
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Problems solved by technology

However, due to the existence of gene transfer, it is difficult to distinguish Bacillus anthracis from other Bacillus cereus even by PCR method, and cross-reaction often occurs.
[0004] In recent years, the inventor's research group found that in the monitoring of fur anthrax, the primers in the PCR diagnostic method recommended by OIE can cross-react with Bacillus cereus and Bacillus thuringiensis, which brings troubles to the diagnosis of anthrax

Method used

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  • Bacillus anthracis duplex fluorescence quantitative PCR detection kit and detection method
  • Bacillus anthracis duplex fluorescence quantitative PCR detection kit and detection method
  • Bacillus anthracis duplex fluorescence quantitative PCR detection kit and detection method

Examples

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Embodiment 1

[0042] Example 1. Design of primers and probes

[0043] The chromosomal gene BA5345 of Bacillus anthracis is a specific identification gene of Bacillus anthracis, which can effectively distinguish the related species in the Bacillus cereus group; the PA gene is a relatively conserved gene on the virulence factor pXO1 plasmid of Bacillus anthracis. In this example, the BA5345 gene and the PA gene were used as targets to design primers and probes.

[0044] In order to detect the Bacillus anthracis chromosomal genome, according to the Bacillus anthracis BA5345 gene (FJ694154.1) registered in GenBank, the primers and probes of the BA5345 gene were designed by using Primer Express3.0.1 software, and the probes of the BA5345 gene were labeled with FAM fluorescein. Needle. The forward primer, reverse primer and probe of the BA5345 gene are as follows:

[0045] BA5345-F: 5'-ATGGGGATACCAGGATGGGT-3',

[0046] BA5345-R: 5'-GCATTACATACCCCAAAGGT-3',

[0047] BA5345-P: 5'-FAM-TCCAAACCCC...

Embodiment 2

[0053] Embodiment 2. The establishment of Bacillus anthracis routine PCR method

[0054] Conventional PCR-specific primers for Bacillus anthracis BA5345 gene and PA gene were designed and synthesized. The specific sequence is:

[0055] BA5345-F: 5'-TGGATTGCGTATGCAGTTCTAC-3',

[0056] BA5345-R: 5'-TTGTAATTCACCATTGAAGCAA-3' and

[0057] PA-F: 5'-GATGTTGCACAATGGTTGATGAA-3',

[0058] PA-R: 5'-GATCCCGTTGGTACTATCC-3'.

[0059] Using the extracted Bacillus anthracis vaccine strain DNA as a template, the BA5345 gene and PA gene were respectively amplified. Add sequentially to the 50 μL reaction system: 5.0 μL of 10×PCR buffer, 1.0 μL of dNTPs (10.0 mmol / L), Mgcl 2 (25mmol / L) 2.0μL, forward and reverse primers (25pmol / μL) 1.0μL each, Taq DNA polymerase (5U / μL) 0.4μL, template DNA 4.0μL, finally add sterilized deionized water to make up to 50.0 μL. Amplification was performed according to the following procedure: 94°C, 3min; 94°C, 30s, 55.0°C, 30s, 72°C, 45s, 35 cycles; 72°C, 10m...

Embodiment 3

[0061] Embodiment 3 positive control is the preparation of plasmid standard

[0062] The BA5345 gene and PA gene fragments amplified in "1.2" were recovered according to the instructions of the gel recovery kit, the target fragments were respectively connected to the pUC57 vector, transformed into E. coli DH5α-competent cells, and positive colonies were picked and identified by PCR and sequencing After correctness, the OD value of the plasmid was measured by NanoDrop2000, converted into copy number by formula, and used as the plasmid standard product of pUC57-BA5345 and pUC57-PA, that is, the positive control for future use.

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Abstract

The invention belongs to the technical field of gene detection, and provides a specific primer and a probe for double fluorescence quantitative PCR detection of Bacillus anthracis, comprising a forward primer BA5345-F and a reverse primer BA5345-R of Bacillus anthracis BA5345 gene and a probe BA5345-P; and a forward primer PA-F, a reverse primer PA-R of Bacillus anthracis PA gene and probe PA-P. The invention also provides a detection kit comprising the primer and the probe, and a method for detecting Bacillus anthracis using a dual fluorescence quantitative PCR. The kit and the method of theinvention can accurately, simply, efficiently and quickly detect Bacillus anthracis.

Description

technical field [0001] The invention belongs to the field of gene detection, and more specifically relates to the detection of fluorescent quantitative PCR of Bacillus anthracis. Background technique [0002] Bacillus anthracis (Bacillus anthracis) is a Gram-positive bacillus that can cause anthrax that is zoonotic. It is an animal disease that must be notified established by the World Organization for Animal Health (OIE). Bacillus anthracis has strong resistance and can exist in animal fur and soil for a long time after forming spores. Bacillus anthracis is highly pathogenic and poses a great threat to the health and life of humans and animals. It is often used as a biological terrorism agent, and its rapid and accurate diagnosis has always been valued. The gold standard for the diagnosis of Bacillus anthracis is the isolation of Bacillus anthracis, but the traditional detection method takes a long time and is difficult to meet the needs of epidemic control. [0003] Baci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/07
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 王素华帅江冰袁淑辉张晓峰蔡军吴绍强吕继洲王建昌孙涛
Owner 王素华
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