Rapid fluorescence PCR detection kit for ASFV (African swine fever virus)

An African swine fever virus and detection kit technology, applied in the field of bioengineering, can solve the problems of complicated operation and long time-consuming, and achieve the effect of reducing manual operation and shortening time.

Pending Publication Date: 2019-04-09
ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological detection methods such as HAD, ELISA, and IFA recommended by OIE are time-consuming

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Primer Design and Construction of African Swine Fever Virus pEASY-T1-p72 Gene Recombination Plasmid

[0022] According to the p72 gene sequence of 24 genotype reference strains of African swine fever virus included in GenBank (see Table 1), screen the conserved region, design primers and probes, primers ASFV-F\ASFV-R and probe ASFV-P are as follows :

[0023] ASFV-F: 5'-ATCCGATCACATTACCTA-3' SEQ ID NO: 3

[0024] ASFV-R: 5'-GTGGTCTTTCAAAGCAAAGG-3' SEQ ID NO: 4

[0025] ASFV-P: 5'-FAM-TCCGTAACTGCTCATGGTATCAAT-3'-BHQ-1 SEQ ID NO:5.

[0026] Table 1 African swine fever reference strains

[0027] Strain name

[0028] Primers were designed according to the p72 gene of the ASFV reference gene type II Georgia2007 strain (accession number: AM999764) registered on GenBank, and the p72 gene was amplified by PCR using it as a template; the reaction system for PCR amplification was 20 μl, and 2×Taq PCR Mix 10μl, primers ASFV-F (20μM) and ASFV-R (20μM) each 1...

Embodiment 2

[0029] Embodiment 2 Establishment of African swine fever virus fluorescent PCR rapid detection reagent reaction system

[0030] The pEASY-T1-p72 strain with correct sequencing results in Example 1 was expanded and cultured, the plasmid was extracted, and fluorescent PCR amplification was performed using it as a template. Reaction system: 2×TransStart Probe qPCR SuperMix 12.5μl, 20μmol / LASFV-F / ASFV-R 0.5μl each, 20μmol / L ASFV-P 0.4μl, then add 1μl pEASY-T1-p72 plasmid DNA as template, add sterilization Double distilled water to bring volume to 25 μl. The PCR reaction program of the reagent is: UNG treatment: 50°C, 2min; pre-denaturation: 95°C for 3min, 1 cycle; pre-amplification: 95°C for 8s, 55°C for 8s, 1 cycle; PCR amplification: 95°C 8s; 8s at 55°C, 40 cycles, collecting fluorescence signals.

Embodiment 3

[0031] Embodiment 3 Establishment of African swine fever virus fluorescent PCR detection kit detection method

[0032](1) For the reaction system of this kit: 17 μl of PCR reaction solution, 3 μl of enzyme mixture, 5 μl of positive control / negative control / sample to be tested, the total volume is 25 μl. The PCR reaction program is: the PCR reaction program of the reagent is: UNG treatment: 50°C, 2min; pre-denaturation: 95°C for 3min, 1 cycle; pre-amplification: 95°C for 8s, 55°C for 8s, 1 cycle; PCR Amplification: 95°C for 8s; 55°C for 8s, 40 cycles, where fluorescent signals are collected.

[0033] (2) Judgment of the test results of this kit: a. Positive: The Ct value of the specimen test result is ≤35 or there is an obvious exponential growth period, and it is determined that the African swine fever virus nucleic acid is detected. b. Suspicious: the Ct value of the test result of the specimen is in the range of 35-38. At this time, the specimen should be tested again. If ...

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PUM

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Abstract

The invention discloses a rapid fluorescence PCR detection kit for ASFV (African swine fever virus). The kit comprises specific primers ASFV-F and ASFV-R as well as a TaqMan probe ASFV-P, the 5' end of the probe labels fluorescence dye as FAM and the 3' end labels a fluorescence quenching group as BHQ-1. The kit can be used for detecting the ASFV in nasal swabs, blood, serum, plasma and tissue ofswine to realize rapid detection of the ASFV. In the whole ASFV detection process, only 40 min is taken from DNA extraction to obtaining of detection results, so that the detection time is greatly shortened, and the detection efficiency is improved.

Description

technical field [0001] The invention relates to a fluorescent PCR detection method for African swine fever virus, in particular to the establishment and application method of a fluorescent PCR rapid detection kit for African swine fever virus, and belongs to the technical field of bioengineering. Background technique [0002] African swine fever (ASF) is a severe, hemorrhagic infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). The acute type is characterized by high fever, reticuloendothelial hemorrhage, and high lethality, with a case fatality rate close to 100%. The World Organization for Animal Health (OIE) lists it as a class of disease that must be declared, which is more harmful to the pig industry. In August 2018, the African swine fever epidemic was reported for the first time in Liaoning Province of my country, which marked the introduction of African swine fever into my country and caused huge losses to our pig industry. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107
Inventor 赵林萍娄亚坤任宝红杨楠侯亚博付燕峰崔兴涛张杰曾小宇
Owner ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD
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