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Primer and probe sequences and method for detecting Bacillus anthracis, Yersinia pestis and legionella pneumophilia through multiple real-time fluorescence polymerase chain reaction (PCR)

A technology of Yersinia pestis, primer-probe sequences, applied in biochemical equipment and methods, determination/inspection of microorganisms, resistance to vector-borne diseases, etc.

Inactive Publication Date: 2011-07-27
中国人民解放军南京军区军事医学研究所
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] At present, there is no primer, probe sequence and method for multiple real-time fluorescent PCR that can simultaneously detect Bacillus anthracis, Yersinia pestis and Legionella pneumophila, and this primer, probe sequence and method are very useful for dealing with emergencies. It is of great significance for the early detection of infectious disease epidemic situation and epidemiological investigation, emergency response to public health emergencies, etc.

Method used

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  • Primer and probe sequences and method for detecting Bacillus anthracis, Yersinia pestis and legionella pneumophilia through multiple real-time fluorescence polymerase chain reaction (PCR)
  • Primer and probe sequences and method for detecting Bacillus anthracis, Yersinia pestis and legionella pneumophilia through multiple real-time fluorescence polymerase chain reaction (PCR)
  • Primer and probe sequences and method for detecting Bacillus anthracis, Yersinia pestis and legionella pneumophilia through multiple real-time fluorescence polymerase chain reaction (PCR)

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Experimental program
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Effect test

preparation example Construction

[0089] 2. Preparation of Target Gene Plasmid Standards

[0090] Extract genomic DNA from inactivated Bacillus anthracis, Yersinia pestis and Legionella pneumophila; A pair, a set of mip primer pairs, and a set of pile primer pairs were amplified by PCR to obtain the corresponding target gene amplification products (capA gene fragment, PA gene fragment, pla gene fragment, caf1 gene fragment, Mip gene fragment , pilE gene fragments); each amplified product was recovered and purified, and respectively connected to PGEM-T easy plasmid, transformed, and extracted plasmid DNA, thereby obtaining the plasmid standard product of each target gene; determining the plasmid standard product of each target gene After the concentration is obtained, dilute and aliquot as needed, and store at -20°C for later use.

[0091] Among them, the methods for extracting genomic DNA include: DNA extraction solution extraction, PCR reaction solution lysis extraction, or DNA extraction kit extraction, all...

Embodiment 1

[0132] Example 1 Triple real-time fluorescent quantitative PCR detection of Bacillus anthracis, Yersinia pestis, and Legionella pneumophila.

[0133] (1) After inactivating Bacillus anthracis, Yersinia pestis and Legionella pneumophila respectively, they were mixed as mock positive samples. Extract the genomic DNA of the simulated positive sample to obtain the test solution.

[0134] (2) Two 25 μl triple real-time fluorescent quantitative PCR reaction systems under the same reaction conditions were used: the first reaction system was directed against the capA gene of Bacillus anthracis, the pla gene of Yersinia pestis, and the Mip gene of Legionella pneumophila; The second reaction system targets the PA gene of Bacillus anthracis, the caf1 gene of Yersinia pestis, and the pilE gene of Legionella pneumophila.

[0135]Both the first and second reaction systems selected primer pairs and probes corresponding to each gene from Table 1, and the primer pairs and probes selected by t...

Embodiment 3

[0161] Example 3 Duplex real-time fluorescent quantitative PCR detection of pla gene and caf1 gene of Yersinia pestis.

[0162] (1) Inactivate Yersinia pestis as a mock positive sample. Extract the genomic DNA of the simulated positive sample to obtain the test solution.

[0163] (2) A reaction system is adopted: the reaction system is aimed at the pla gene and caf1 gene of Yersinia pestis. The reaction system selects primer pairs and probes corresponding to each gene from Table 1. The primer pairs and probes selected by it consist of two sets of primer pairs and two probes: a set of pla primer pairs and a pla probe , and a set of f1 primer pairs and a f1 probe. For example: select primer pairs: pla-F and pla-R, and f1-F and f1-R; select probes: pla-probe and f1-probe. It should be pointed out that the primer pairs and probes selected in the reaction system can be any set of primer pairs and any probes for the aforementioned genes.

[0164] Labeling of probes: the 5' end o...

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Abstract

The invention relates to primer and probe sequences and a method for detecting Bacillus anthracis, Yersinia pestis and legionella pneumophilia through multiple real-time fluorescence polymerase chain reaction (PCR). The primer and probe sequences comprise a set of sequences of primers and probes for the Bacillus anthracis, Yersinia pestis and legionella pneumophilia; and the method comprises the following steps of: selecting primer pairs and probes; preparing a plasmid standard of a target gene; establishing a reaction system; and selecting a fluorescent detection channel. The primer pairs and probes have good specificity and performance, and a multiple real-time fluorescence PCR method for detecting the Bacillus anthracis, Yersinia pestis and legionella pneumophilia can be established, and has the advantages of reliable and accurate results, sensitivity, simple operation, time and labor conservation, low cost, high efficiency and the like.

Description

technical field [0001] The present invention relates to a PCR primer, probe sequence and method for detecting pathogenic microorganisms, especially a primer, probe sequence and method for simultaneously detecting multiple real-time fluorescent PCR of Bacillus anthracis, Yersinia pestis and Legionella pneumophila, Belongs to the field of biotechnology. Background technique [0002] Bacillus anthracis can cause anthrax in sheep, cattle, horses and other animals and humans, seriously threatening public property, health and life safety. Yersinia pestis can cause severe infectious disease plague, the disease spreads quickly, and the fatality rate is high (70%-100%). There have been many devastating epidemics in history, and it is also a classic lethal bacterium. Legionella pneumophila can cause Legionnaires' disease mediated by aerosols, and the epidemic fatality rate can be as high as 30%. Since the first confirmed case of Legionella in Nanjing in 1982 in my country, there have...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 王长军张锦海邓小昭王忠灿
Owner 中国人民解放军南京军区军事医学研究所
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