African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, African swine fever fluorescent PCR assay kit and application thereof

A detection kit and African swine fever technology, applied in the field of bioengineering, can solve the problems of unsuitable infection and early use of serological diagnostic methods, and achieve the effect of reducing manual operation, high sensitivity and strong specificity

Active Publication Date: 2017-07-18
CHINESE ACAD OF INSPECTION & QUARANTINE
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Problems solved by technology

Serological diagnostic methods are not suitable for early use in infection due to the time required for antibody production

Method used

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  • African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, African swine fever fluorescent PCR assay kit and application thereof
  • African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, African swine fever fluorescent PCR assay kit and application thereof
  • African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, African swine fever fluorescent PCR assay kit and application thereof

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Embodiment

[0030] Materials and reagents used in the present invention: inactivated cell culture virus liquid, African swine fever pseudovirus sample (5×10 6 TCID 50 ).

[0031] DNeasy Tissue (QIAGEN) Animal Tissue Extraction DNA Kit, DNA Amplification Basic Kit (TaKaRa), pGEM-T Vector System Kit (Promega), Agarose Gel DNA Purification Kit, DNA Marker, etc.

[0032] Instruments used in the present invention:

[0033] iCycler iQ TM 5 Fluorescence real-time multi-wavelength PCR detection system, PTC-200 gradient PCR instrument, ALPHA-5500 gel imaging system, etc.

[0034] Primer design and synthesis

[0035] In this study, according to the ASFV p72 (GeneBank No. AY578689) gene sequence, the primer design software Primer5.0 was used to design the amplification primers.

[0036] p72U:5'-TATGGCATCAGGAGGAGC-3' (SEQ ID NO:4)

[0037] p72L:5'-GGTACTGTAACGCAGCAC-3', (SEQ ID NO:5)

[0038] The length of the amplified product was 1939bp.

[0039] Fluorescent PCR-specific primers and probes ...

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Abstract

The invention relates to an African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, an African swine fever fluorescent PCR assay kit and application thereof, which belong to the field of bioengineering. With the p72 gene as a reference, a pair of specific PCR primers and a TaqMan fluorescent probe are designed and optimized. Moreover, the primers are designed with the full length of the p72 gene to amplify the p72 gene, and the PCR product is cloned into a pGEM-T vector. Standard curves are drawn with a positive plasmid as a standard product, and the assay range is 10 to 1.0*10<7> copies. In the whole process of assaying African swine fever by an African swine fever fluorescent PCR method disclosed by the invention, only 2.5 to 3 hours are taken from DNA extraction to the appearance of an assay result, manual operation is greatly reduced, and time is greatly shortened. Furthermore, a plurality of samples can be assayed each time, and particularly assay of a large batch of samples can be realized.

Description

technical field [0001] The invention belongs to the field of biological engineering, and in particular relates to a fluorescent PCR detection reagent for African swine fever, a fluorescent PCR detection kit for African swine fever and applications thereof. Background technique [0002] African swine fever (ASF) is a severe, hemorrhagic infectious disease of pigs caused by African swine fever virus (ASFV), with a case fatality rate close to 100%. Montgomery was first discovered in Kenya, Africa in 1921. Because it is harmful to the pig industry, and there is no effective vaccine control, it is listed as the first of the legally reported infectious diseases of pigs by OIE, and is highly concerned by countries all over the world (Donald P.King et al., 2003; Chang Hua et al., 2007). Since ASFV has a complex immune evasion mechanism, lacks typical neutralizing antibodies, and has no effective vaccine for epidemic prevention (Sun Huaichang, 1999), it is particularly important to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2561/101
Inventor 邓俊花刘晗冯春燕林祥梅吴绍强
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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