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Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild strains

A technology of African swine fever virus and primer probes, applied in the field of real-time fluorescent PCR primer probe combinations and kits, can solve the problems of interference with attenuated vaccines, unfavorable control, inability to distinguish wild-type strains and vaccine viruses, etc.

Active Publication Date: 2020-02-07
广纳达康(广州)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the African swine fever virus MGF360-505R deletion attenuated vaccine strain has shown good safety and protective effects; however, once the vaccine is applied clinically, the existing detection technology based on the African swine fever virus P72 gene will not be able to Distinguish between wild-type strain virus and vaccine virus, resulting in positive detection of both vaccine virus and wild-type virus
This will greatly interfere with the detection of African swine fever virus wild virus after the attenuated vaccine is used, which is not conducive to the control of the disease

Method used

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  • Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild strains
  • Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild strains
  • Real-time fluorescent PCR primer probe combination and kit for detecting African swine fever virus wild strains

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Screening of real-time fluorescent PCR primer-probe combinations for detection of African swine fever virus wild virus

[0044] According to the genome target sequence missing from the attenuated vaccine, that is, the 27942-35500bp sequence of the African swine fever virus Chinese epidemic strain Pig / CN / HLJ / 2018, considering the mismatch of primers and Tm values, the designed primers and probes Analyzed in the NCBI database by Blast software, the primer pairs and probe sequences must cover all known African swine fever virus sequences in the database, and have no obvious pairing with other sequences. Through the analysis, it was found that:

[0045]The sequences 5'-TCTGCGTCAACTACCTCG-3', 5'-TAAACTACTCCGTGAAAC-3' and 5'-GACGAAAGTGAGGACGAT-3' can match the sequences of most African swine fever viruses, and can be used as a combination for detecting wild African swine fever viruses, that is, primers Pair (upstream primer: 5'-TCTGCGTCAACTACCTCG-3' and downstream primer: 5'...

Embodiment 2

[0059] The kit for detecting the wild-type strain of African swine fever virus includes the above-mentioned fluorescent PCR primer-probe combination; in the present invention, the primers and probes can be directly added to the commercially available qPCR Probe MasterMix reagent. The method for detecting and detecting African swine fever virus wild virus based on above-mentioned kit construction comprises the following steps:

[0060] 1) Preparation of standard products:

[0061] Using African swine fever virus genomic DNA as a template, use fluorescent PCR primers:

[0062] Upstream primer: 5'-TCTGCGTCAACTACCYCG-3',

[0063] Downstream primer: 5'-ATMGTCYTYACTTTCRTC-3', Y is C / T; M is A / C, R is A / G,

[0064] Perform PCR amplification to obtain a 258bp gene partial fragment (as shown in SEQ ID NO: 4), which is connected to the pMD18-T vector to construct a positive plasmid pMGF_505-2R containing the target fragment, and the sequencing result has no mutation;

[0065] 2) Esta...

Embodiment 3

[0072] The process flow and result judgment of the detection method of the kit for detecting the African swine fever virus wild-type strain ( image 3 ), including the following steps:

[0073] (1) Take the sample to be tested and extract the genome

[0074] (2) Detection by fluorescent PCR:

[0075] The fluorescent PCR reaction system is Mix 5 μL, ROX 0.2 μL, 10 μM fluorescent PCR primers 0.25 μL each, 10 μM fluorescent PCR probes 0.1 μL, DNA template 2 μL, water up to 10 μL;

[0076] Fluorescence PCR reaction conditions: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, annealing at 50°C for 10s, extension at 60°C for 30s, 45 cycles, and fluorescence signals were collected at 60°C;

[0077] (3) Judgment of test results:

[0078] If Ct=40 or no amplification curve, it indicates that the sample does not contain wild virus or the content of wild virus is so low that it cannot be detected; further detection: based on p72 If the fluorescent PCR is positive, it i...

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Abstract

The invention relates to the technical field of animal virus detection in the field of veterinarians, and particularly discloses a real-time fluorescent PCR primer probe combination and a kit for detecting African swine fever virus wild strains. A PCR method based on the kit can not only specifically detect the African swine fever virus wild strains, but also serve as a method for distinguishing between the African swine fever virus wild strains and MGF360-505R loss attenuated vaccine virus strains when in use in combination with current p72 fluorescent PCR, therefore, important tools are provided for detecting the African swine fever virus wild strains and animals infected by the African swine fever virus wild strains, and control over African swine fevers and purification of the Africanswine fevers are promoted.

Description

technical field [0001] The invention relates to the technical field of animal virus detection in the veterinary field, in particular to a real-time fluorescent PCR primer-probe combination and a kit for detecting African swine fever virus wild virus. Background technique [0002] African swine fever (ASF) is a virulent, hemorrhagic infectious disease of pigs caused by African swine fever virus (ASFV); the virulent strain can be fatal within about 5-14 days of infection Pigs, of which the mortality rate is close to 100%, and are difficult to extinguish. The disease is a statutory reportable animal disease stipulated by the World Organization for Animal Health (OIE). Since the first African swine fever epidemic occurred in my country in August 2018, it has dealt a very heavy blow to my country's pig industry. [0003] Since the discovery of African swine fever virus more than 100 years ago, only a handful of countries such as Spain and Brazil have successfully controlled Afr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 张安定谭臣
Owner 广纳达康(广州)生物科技有限公司
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