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RPA (recombinase polymerase amplification) primer and detection kit for rapidly detecting African swine fever viruses

A technology of African swine fever virus and kit, which is applied in the field of preventive veterinary inspection, can solve the problems of being expensive and difficult to meet the detection requirements, and achieve the effect of simple operation, high specificity and good specificity

Pending Publication Date: 2019-05-24
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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Problems solved by technology

[0003] At present, the detection methods for African swine fever virus mainly include molecular biology methods such as PCR and fluorescent quantitative PCR. Testing requirements

Method used

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  • RPA (recombinase polymerase amplification) primer and detection kit for rapidly detecting African swine fever viruses
  • RPA (recombinase polymerase amplification) primer and detection kit for rapidly detecting African swine fever viruses
  • RPA (recombinase polymerase amplification) primer and detection kit for rapidly detecting African swine fever viruses

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Embodiment 1

[0046] 1. Establishment of RPA detection primers, probes and detection methods for African swine fever virus

[0047] 1. Design and synthesis of RPA primers and probes for African swine fever virus

[0048] For the African swine fever virus p72 gene from GenBank, according to the RPA primer and probe design principles, the primers and probes are designed as follows (Table 1).

[0049] The above primers and probes were all synthesized.

[0050] Table 1 Primers and probes used in RPA:

[0051]

[0052] 2. Establishment of ASFV RPA gel detection system and primer screening:

[0053] The genome extracted by the magnetic bead extraction kit was used as a template for amplification, and the designed primers were screened to screen out the optimal primer pair:

[0054] ① Use F as the upstream primer to screen the downstream primers.

[0055] After the reaction, use 2% agarose electrophoresis to identify the best downstream primers. Test results such as figure 2 As shown in ...

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Abstract

The invention discloses an RPA (recombinase polymerase amplification) primer and a detection kit for rapidly detecting African swine fever viruses. Target genes can be effectively amplified, specificity is 100%, detection sensitivity is 102 copy / reaction, and sensitivity is equivalent to that of fluorescent quantitative PCR (polymerase chain reaction). Cross reaction between the RPA amplificationprimer and one of classical swine fever viruses, vesicular exanthema swine viruses I, porcine reproductive and respiratory syndrome viruses, porcine circoviruses and the like is omitted. A RPA isothermal amplification system is rapidness in reaction and wide in temperature range, effective amplification of the target genes can be achieved at the temperature of 38-46 DEG C, and the detection kit can rapidly, efficiently and sensitively detect the African swine fever viruses and has the advantages that the kit is simple to operate, high in specificity, safe and free from pollution, reaction results are easily observed and the like. Effective technical means are provided for on-site rapidness detecting and screening of infection nucleic acid of the African swine fever viruses, and the RPA amplification primer has great significance for control of infection spreading of the African swine fever viruses in China and inspection and quarantine in infected areas and entry and exit ports.

Description

Technical field: [0001] The invention relates to a nucleic acid detection kit for detecting African swine fever virus, and also provides RPA amplification primers for detecting African swine fever virus, belonging to the field of preventive veterinary testing. Background technique: [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease caused by African swine fever virus (ASFV) infection, which has a devastating blow to the development of the breeding industry, mainly causing High fever, vomiting, constipation, pink or purple skin on the surface of the body, splenomegaly, darkened color, and brittle texture can be seen at autopsy. [0003] At present, the detection methods for African swine fever virus mainly include molecular biology methods such as PCR and fluorescent quantitative PCR. Testing requirements. Contents of the invention [0004] The invention provides an RPA nucleic acid test strip kit for detecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/11C12R1/93
Inventor 缪发明扈荣良张守峰张静远李楠陈腾张菲
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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