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Recombinase polymerase application (RPA) primer for rapidly detecting African swine fever virus (ASFV) nucleic acid, preparation method of RPA primer, and kit

An African swine fever virus, fast technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as expensive and unsuitable for general laboratory promotion and application, and achieve high specificity and sensitivity strong effect

Inactive Publication Date: 2018-04-20
SHIHEZI UNIVERSITY
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Problems solved by technology

However, the above methods either require expensive instruments and equipment, or require skilled technicians, or need to design complex primers and probes, or the reaction reagents need to be transported and stored in a cold chain, and are not suitable for general laboratory application.

Method used

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  • Recombinase polymerase application (RPA) primer for rapidly detecting African swine fever virus (ASFV) nucleic acid, preparation method of RPA primer, and kit
  • Recombinase polymerase application (RPA) primer for rapidly detecting African swine fever virus (ASFV) nucleic acid, preparation method of RPA primer, and kit
  • Recombinase polymerase application (RPA) primer for rapidly detecting African swine fever virus (ASFV) nucleic acid, preparation method of RPA primer, and kit

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Embodiment Construction

[0055] 1. Synthesis of ASFV P72 gene and construction of its recombinant plasmid

[0056] According to the ASFV genome reference sequence registered in GenBank (accession number: AY578706), the P72 gene sequence (1941bp) was determined, and the P72 full-length cDNA (1941bp) was synthesized in vitro, and cloned into the pMD-18T vector to obtain the recombinant plasmid pT-ASFV -P72. The pT-ASFV-P72 plasmid was transformed into competent cells DH5α, and the constructed recombinant plasmid was verified by PCR and sequencing. A specific band can be obtained as a result of PCR amplification; the sequencing result of the amplified product shows that the homology of the sequence with the ASFV P72 gene (AY578706) is 100%. After the recombinant positive bacteria containing the recombinant plasmid were cultivated overnight, the plasmid was extracted and its concentration was determined. It was determined that the concentration of the recombinant plasmid pT-ASFV-P72 was 52ng / μL.

[005...

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Abstract

The invention discloses a recombinase polymerase application (RPA) primer for rapidly detecting African swine fever virus (ASFV) nucleic acid, a preparation method of the RPA primer, and a kit, belonging to the technical field of biology. A pair of RPA primers, i.e., an upstream primer <210>2 and a downstream primer <210>3 which have high specificity and strong sensibility are screened out; an RPAdetection system for rapidly detecting the ASFV nucleic acid is further established by means of the primers. Compared with the common polymerase chain reaction (PCR) method, RPA-lateral flow assay (LFA) has the advantages that firstly, the RPA-LFA belongs to an isothermal amplification technology and is low in requirements for instruments and equipment, and reactions can be completed only by means of a constant temperature water bath kettle; secondly, the detection speed of the RPA-LFA is fast, and the reaction time is 40 minutes and is shorter than the conventional PCR reaction time; thirdly, the visualization of detection results can be realized. Due to the characteristics, the RPA-LFA method established by the invention can be used for rapid detection and differential diagnosis of ASFVin common laboratories of grassroots units.

Description

technical field [0001] The invention relates to an RPA primer for rapidly detecting African swine fever virus nucleic acid, a preparation method and a kit, belonging to the field of biotechnology. Background technique [0002] African swine fever (African swine fever, ASF), also known as African swine fever, is an acute and highly contagious infectious disease that seriously harms pigs and wild boars caused by African swine fever virus (ASFV) infection. The disease course is short, and the mortality rate is as high as 100%. It is one of the most serious severe infectious diseases to the world's pig industry. The disease belongs to the animal epidemic disease required to be reported by the World Organization for Animal Health (OIE), and ASFV is listed as a first-class animal pathogenic microorganism in the list of animal pathogenic microorganisms in my country. my country is one of the big pig-raising countries dominated by agriculture. Although there is no report of this di...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 乔军孟庆玲李杰李静郭晶田路路乔梦凡孟丹伍晔晖李重阳田振中张星星蔡扩军张再超赵春光
Owner SHIHEZI UNIVERSITY
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