Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method

A Brucella, fluorescence quantitative technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of many influencing factors, poor method sensitivity, easy cross-contamination, etc. Specificity, ease of operation, and the effect of shortening detection time

Inactive Publication Date: 2011-08-10
浙江国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods generally have shortcomings such as long detection time, cumbersome operation, poor sensitivity of the method, and many influencing factors, which cannot meet the needs of fast and accurate
The discovery of traditi

Method used

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  • Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method
  • Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method
  • Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method

Examples

Experimental program
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Example Embodiment

[0018] Example 1 Preparation of primers and probes

[0019] 1. Target gene selection

[0020] The Brucella cell surface protein 31kD (Brucella Cell Surfacial Protein, BCSP31) exists on the cell surface of the bacteria. This protein is encoded by the BCSP31 gene, which is species-specific and highly conserved. It can be used as a nucleic acid diagnostic marker for Brucella According to the genbank database search, the gene sequence was obtained.

[0021] 2. Design and screening of PCR primers

[0022] Following the principle of composite probe design and based on the BCSP31 gene sequence, two sets of primers and probes were designed.

[0023] The 5'end of the fluorescent probe is labeled with a fluorescent molecule FAM as a reporter group, and the 3'end is labeled with phosphoric acid to block its extension. A quenching group Dabcyl is attached to the 3'end of the quenching probe.

[0024] In order to screen out a combination with high amplification efficiency from the two sets of compo...

Example Embodiment

[0035] Example 2. Detection of Brucella

[0036] In order to investigate the practical application ability of the detection of the present invention, a blood simulation specimen contaminated by Brucella was specially prepared, and at the same time, a pure bacteria was set as a positive control, and a non-brucella pathogenic bacteria was used as a negative control.

[0037] 1. Sample Preparation

[0038] (1). Preparation and treatment of contaminated blood: Brucella fixed value is serially diluted with anticoagulant blood to make a concentration of 1×10 6 CFU / ml-1×10 1 CFU / ml contaminated blood sample. Take 1ml of blood containing different concentrations of bacteria, centrifuge at 12000rpm for 10 minutes, discard the supernatant, and add 1ml of red blood cell lysate (50mmol / L TrisHCL, 25mmol / L KCl, 5mmol / LMgCl) 2 , PH7.5, TKM solution), shake vigorously for 2 minutes, centrifuge at 12000 rpm for 10 minutes to discard the supernatant, add 1 ml of TKM solution, mix, and centrifuge at 1...

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Abstract

The invention provides a reagent for detecting brucella. The reagent comprises an upstream primer, a downstream primer, a fluorescence probe and a quenching probe; the gene sequence of the upstream primer is 5'-caagggcaaggtggaagatt-3'; the gene sequence of the downstream primer is 5'-ctgcgaccgatttgatgttt-3'; the gene sequence of the fluorescence probe is 5'-fam-atcgtttccgggtaaagcgtcgcca-P-3'; and the gene sequence of the quenching probe is 5'-cgctttacccggaaacga-Dabcyl-3'. The invention also provides a complex probe fluorescence quantitative polymerase chain reaction (PCR) brucella detection method using the reagent. The method is simple and convenient in operation, efficient, quick and specific; the detection time of the brucella is greatly shortened; the quantitative detection of a sample can be completed in about 2 hours; and the reagent and the method have significance for early diagnosis of brucella disease.

Description

technical field [0001] The invention relates to a reagent for detecting brucella and a detection method for brucella, in particular to a method for amplifying a composite probe brucella marker gene and then using fluorescent PCR technology to detect brucella. Background technique [0002] Brucellosis (brucellosis) is a zoonotic natural foci infectious disease caused by Brucella. Brucella can infect humans, a variety of domestic and wild animals, and cause fever, abortion and infertility, chronic arthritis, and neurological damage. After human beings are infected with brucellosis, due to the long course of the disease, it will cause huge economic losses and serious public health problems. In recent years, due to the gradual increase in livestock breeding and the increasingly frequent product circulation, brucellosis epidemics in humans and livestock have rebounded sharply in a considerable range. [0003] The most critical technical link in the prevention and control of inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12N15/11G01N21/64
Inventor 吴忠华吕沁风郑伟黄建可李禾
Owner 浙江国际旅行卫生保健中心
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