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Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis

A Yersinia, fluorescent quantitative technology, applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of easy cross-contamination, poor sensitivity of the method, and incapable of quantitative analysis, etc., to achieve Guaranteed specificity and easy operation

Inactive Publication Date: 2011-08-10
浙江国际旅行卫生保健中心
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Problems solved by technology

These methods have been used for decades and have played an important role in bacterial identification, but there are generally deficiencies such as detection time lag and poor sensitivity of the method, which cannot meet the needs of rapid and accurate
The discovery of traditional PCR technology and nucleic acid hybridization technology has greatly promoted the development of bacterial detection technology, but there are shortcomings such as easy cross-contamination and incapable of quantitative analysis.

Method used

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  • Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis
  • Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis
  • Reagent for detecting Yersinia pestis and method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis

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Embodiment 1

[0023] The preparation of embodiment 1 primer and probe

[0024] 1. Target gene selection

[0025] The Yersinia pestis 6MD plasmid has the pst gene that produces Yersinia pestis and the gene of plasma coagulase and plasminogen activator pla, which is considered to be related to human pathogenicity and can be used as the gene of Yersinia pestis Diagnostic sign. The pla gene sequence was obtained by searching the genbank database.

[0026] 2. Design and screening of fluorescent probes, quencher probes and PCR primers

[0027] According to the principle of compound probe design, two sets of primers and probes were designed according to the sequence of pla gene. The 5' end of the fluorescent probe is labeled with fluorescent molecule FAM as a reporter group, and the 3' end is labeled with phosphate to block its extension. A quenching group Dabcyl is connected to the 3' end of the quenching probe.

[0028] In order to screen out a combination with high amplification efficiency...

Embodiment 2

[0055] Embodiment 2, the detection of Yersinia pestis

[0056] In order to investigate the actual application ability of the detection of the present invention, simulated samples such as blood, water samples, soil, and surface stains contaminated by Yersinia pestis were specially prepared, and pure bacteria were set as positive controls, and non-plague pathogenic bacteria were used as negative controls. .

[0057] 1. Sample Preparation

[0058] (1).Preparation and processing of contaminated blood: fixed-value Yersinia pestis was serially diluted with anticoagulant blood to make a concentration of 1×10 6 CFU / ml-1×10 1 CFU / ml of contaminated blood samples. Take 1ml of blood containing different concentrations of bacteria, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1ml of red blood cell lysate (50mmol / LTrisHCL, 25mmol / L KCl, 5mmol / LMgCl 2 , pH7.5, TKM solution), vigorously shake and mix for 2 minutes, centrifuge at 12000rpm for 10 minutes, discard the...

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Abstract

The invention provides a reagent for detecting Yersinia pestis, which comprises an upstream primer, a downstream primer, a fluorescent probe and a quenching probe. The invention also provides a method for carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) detection on Yersinia pestis by utilizing the reagent. The primer and the probe are designed according a specific pathogenic gene-plasma coagulase and plasmin original activating factor pla gene on a Yersinia pestis 6MD plasmid; and by carrying out BLAST retrieval with the whole genebank database, the reagent is specific only on a pla gene sequence on Yersinia pestis, has no isogeny with nucleotide sequences of other species and ensures the specificity of the detecting method. The method can realize the amplification and synchronous detection of DNA in the same pipe without carrying out gel electrophoresis analysis after PCR, can finish quantitative detection on a sample in 2 hours or so and has the advantages of simple and convenient operation, high efficiency, high speed and specificity.

Description

technical field [0001] The invention relates to a reagent for detecting Yersinia pestis and a method for detecting the plague pathogen, in particular to a method for amplifying the identification gene of the plague pathogen with a compound probe and then detecting the plague pathogen by using a fluorescent PCR technique. Background technique [0002] Plague is a severe infectious disease caused by Yersinia pestis, which seriously endangers human beings. Plague has the characteristics of strong infectivity, rapid spread, severe illness, and high fatality rate, and is one of the international quarantine infectious diseases. The "Law on the Prevention and Control of Infectious Diseases" promulgated by our country also defines plague as a Class A infectious disease. [0003] The most critical technical link in the prevention and control of infectious diseases is to establish laboratory diagnostic methods for pathogen detection and detection, which can accurately and quickly det...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12Q1/06G01N21/64
Inventor 李禾吕沁风吴忠华郑伟
Owner 浙江国际旅行卫生保健中心
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