PCR primer and probe for detecting infectivity of African swine fever virus, kit and detection method

A technology of African swine fever virus and infectivity is applied in the field of PCR primers and probes, kits and detection for rapid detection of African swine fever virus infectivity. High specificity, strong specificity and anti-interference, and simple result judgment

Pending Publication Date: 2020-03-20
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method (PMA-qPCR) has been well applied in differentiating and detecting the infectivity of living and dead bacteria

Method used

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  • PCR primer and probe for detecting infectivity of African swine fever virus, kit and detection method
  • PCR primer and probe for detecting infectivity of African swine fever virus, kit and detection method
  • PCR primer and probe for detecting infectivity of African swine fever virus, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, establishment of African swine fever virus rapid fluorescent quantitative PCR detection method

[0036] 1. Establishment of a rapid fluorescent quantitative PCR detection method for African swine fever virus

[0037] 1. Design and screening of PCR primers and probes for detection of ASFV signature sequence p72 nucleic acid copy number

[0038] The present application designs two specific primers and probes according to the conserved and stable sequence of the B646L gene (p72 protein gene) in the African swine fever virus genome. The specific method is: download all nucleic acid sequences of the ASFV B646L (p72) gene (as shown in SEQ ID NO.4) from NCBI GenBank, perform homologous comparison analysis, and search for the conserved sequence of the p72 gene. In the p72 conserved nucleic acid sequence region, specific primers and target probes were designed. The primers used in this embodiment to detect the conserved nucleic acid sequence of the specific targe...

Embodiment 2

[0060] Example 2: Optimization of PMA-qPCR detection method for African swine fever virus infectivity

[0061] The present invention adopts PMA to pretreat the sample to be tested, through which the exposed DNA can be stably cross-linked so that no PCR reaction can occur, thereby removing the interference caused by the exposed DNA, and achieving the purpose of detecting whether the sample contains infectious ASFV. The specific detection principle of PMA-qPCR is as follows: Figure 6 shown. In order to illustrate the feasibility of this method and optimize the detection conditions, one of the African swine fever virus inactivation methods recommended by the World Organization for Animal Health (OIE) is used to heat to optimize the PMA concentration and photolysis time, so as to determine the PMA pretreatment condition. This example should not be construed as detecting only heat-inactivated ASFV. Based on similar principles and optimization, it is also within the scope of the...

Embodiment 3

[0063] Example 3: PMA-qPCR detection of African swine fever virus in heat-inactivated pig positive blood samples

[0064] Select a porcine whole blood sample positive for p72 nucleic acid, pipette an appropriate amount into several portions, heat inactivate the blood sample at 70°C for 30 minutes, and centrifuge at 10,000 g for 10 minutes to collect the supernatant as the test sample. At the same time, the sample without heat inactivation was used as the control, and the test sample was pretreated with PMA (50 μM) using the optimized PMA-qPCR conditions, and the copy number of p72 was detected after the nucleic acid was extracted respectively. The group without PMA pretreatment was used as a reference. Compared with the control, the group with the same volume of sterilized water as the template was used as the negative control. The results are shown in Table 3.

[0065] Table 3 Ct values ​​of ASFVp72 in heat-treated pig positive blood samples detected by PMA-qPCR

[0066] ...

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Abstract

The invention provides a PCR primer and probe for detecting infectivity of an African swine fever virus, a kit and a detection method. Primer sequences are shown in SEQ ID No. 1~2, and a probe sequence is shown in SEQ ID No. 3. The kit provided by the invention contains the PCR primer and probe and a nucleic acid dye, which is bonded to nucleic acid and has photosensitive characteristics; and whether the infective African swine fever virus is present in a sample to be detected or not is rapidly judged through pretreating the sample to be detected with the photosensitive nucleic acid dye, taking a sample free of photosensitive dye pretreatment as a control sample, then, extracting nucleic acids from the sample to be detected and the control sample, detecting African swine fever virus conserved gene copy numbers of the sample to be detected and the control sample separately by a qPCR method, and comparing the gene copy numbers of the sample to be detected and the control sample. The kitis short in detection consumed time, high in specificity, reliable in result and simple in result judgment, is applicable to any laboratory, prevention and control units and veterinary stations of alllevels of grass roots, large-, medium- and small-size farms and the like and is of far reaching importance in judgment on measures such as killing, prevention and control on the African swine fever virus.

Description

technical field [0001] The invention relates to the field of African swine fever virus detection, more specifically, to a PCR primer, a probe, a kit and a detection method for rapidly detecting the infectivity of the African swine fever virus. Background technique [0002] African swine fever virus (African swine fever virus, ASFV) is a double-stranded DNA virus that infects domestic pigs and wild boars and causes acute hemorrhagic severe infectious disease viruses in pigs. The diseases caused by the virus are collectively referred to as African swine fever (African swine fever) Swine fever, ASF). On August 3, 2018, ASFV first broke out in Shenbei New District, Shenyang City, Liaoning Province, my country, and spread widely across the country a few months later. ASFV has the characteristics of acute infection and high fatality rate, so it has caused great losses to the agricultural economy of China and the world. ASFV has been present in Kenya since 1921, but no effective ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 危宏平刘欢余军平熊进
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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