Recombinase polymerase amplification (RPA) primers and method used for detecting bacillus anthracis

A technology for Bacillus anthracis and purpose, applied in the field of RPA primers for detecting Bacillus anthracis, can solve the problems of difficulty in adapting to rapid diagnosis and control of epidemic situation, long time consumption, etc., and achieve the effects of reliable results, simple operation and rapid response.

Inactive Publication Date: 2020-03-06
王素华
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Problems solved by technology

At present, the commonly used methods for the detection of Bacillus anthracis mainly include culturing the pathogen, staining microscopy, and serological (antigen or antibody) detection, etc., but these detection methods are time-consuming and difficult to meet the needs of rapid diagnosis and control of the epidemic.

Method used

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  • Recombinase polymerase amplification (RPA) primers and method used for detecting bacillus anthracis
  • Recombinase polymerase amplification (RPA) primers and method used for detecting bacillus anthracis
  • Recombinase polymerase amplification (RPA) primers and method used for detecting bacillus anthracis

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Embodiment 1

[0056] 1 Materials and methods

[0057] 1.1 Bacterial strains and clinical samples

[0058] Bacillus anthracis vaccine strain (A16R) was purchased from Lanzhou Biological Products Co., Ltd; Culture Collection Center; Proteus, Staphylococcus aureus and Bacillus subtilis were donated by Hangzhou Customs Technical Center. Genomic DNA of Bacillus anthracis is preserved by our laboratory. The clinical samples are laboratory-made contaminated raw fur samples.

[0059] 1.2 Main reagents

[0060] Quick-DNA TM Extraction and purification kits were purchased from ZYMO RESEARCH, USA; plasmid DNA mini-extraction kit, pUC57 Vector, Escherichia coli DH5α and Amp were purchased from Shanghai Sangon Bioengineering Co., Ltd.; TwistAmp TM DNA amplification Kit was purchased from Twist Dx Company in the UK; Taq DNA polymerase (5U / μL), dNTPs (10.0mmol / L), MgCl 2 (25mmol / L), DL500 DAN Marker, DL5000 DAN Marker, and DNA gel recovery kit were purchased from Bao Biological Engineering (Dalian)...

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Abstract

The invention relates to the technical field of biology, and specifically discloses recombinase polymerase amplification (RPA) primers and a method used for detecting bacillus anthracis. According tothe RPA primers used for detecting the bacillus anthracis, 1 pair of RPA primers are designed and synthesized on basis of a conservative sequence of a bacillus anthracis BA5345 gene; and the RPA primers are shown as SEQ ID NO. 1-2. Subjected to further optimization on primer concentration, reaction temperature and reaction time, the bacillus anthracis can be specifically detected after addition of2.0 microliters of the upstream primer and the downstream primer, of which the concentration is 10 mmol / L, into 50 microliters of a RPA reaction system and subsequent constant-temperature reaction ina water bath pot at 39 DEG C for 30 minutes; and more RPA amplification products can be obtained. Thus, the RPA detection method developed by the invention has an sensitivity which is 100 times higher than sensitivities of conventional PCR methods, as well as a positive detection rate of clinical samples of contaminated fur up to 65.71% which is significantly higher than positive detection rate of the conventional PCR methods. The RPA method established by the invention is easy to operate, fast to react, as well as true and reliable in results; and thus, the RPA method is suitable for rapid detection of the bacillus anthracis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RPA primer and a method for detecting 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 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 detection has always been valued. At present, the commonly used methods for the detection of Bacillus anthracis mainly include culturing the pathogen, staining microscopy, and serological (antigen or antibody) detection, etc., but these detection methods are time-consuming and difficult to meet the needs of rapid diagnosis and epidemic control. [0003] Recombinase polymerase amplification techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/07
CPCC12Q1/689C12Q1/6844C12Q2527/143C12Q2527/101C12Q2527/113
Inventor 王素华曾若雪帅江冰吴绍强王建昌袁淑辉蔡军
Owner 王素华
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