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Real-time fluorescent PCR amplification primer pair and probe primer for identifying and detecting African swine fever virus, and kit prepared from real-time fluorescent PCR amplification primer pair and probe primer

A technology of African swine fever virus and real-time fluorescence, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., to achieve high sensitivity and strong specificity

Active Publication Date: 2021-05-25
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of effective vaccines has become a possible means to prevent and control the epidemic. Given that the current inactivated whole virus vaccines cannot cause effective immune responses, gene-deleted live vaccines have become another possible choice. , there is an urgent need to provide a simple and accurate detection method and its corresponding detection substances in the prior art

Method used

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  • Real-time fluorescent PCR amplification primer pair and probe primer for identifying and detecting African swine fever virus, and kit prepared from real-time fluorescent PCR amplification primer pair and probe primer
  • Real-time fluorescent PCR amplification primer pair and probe primer for identifying and detecting African swine fever virus, and kit prepared from real-time fluorescent PCR amplification primer pair and probe primer
  • Real-time fluorescent PCR amplification primer pair and probe primer for identifying and detecting African swine fever virus, and kit prepared from real-time fluorescent PCR amplification primer pair and probe primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Primer and probe of embodiment 1 African swine fever virus real-time fluorescence

[0043] See sequence table 1 for three pairs of primers and three probes used in the present invention.

[0044] Table 1 Primers and probes for real-time fluorescence of African swine fever virus

[0045]

[0046]

[0047] The reaction system of the African swine fever virus PCR method is shown in Table 2, and the reaction conditions are shown in Table 3.

[0048] Table 2 The reaction system of the African swine fever virus multiplex real-time fluorescent PCR method

[0049] components Volume (μl) 5U / μl Taq DNA polymerase 2 5×reaction buffer 50mM (5×Tris-HCl) 10 2.5mM dNTPs 5 Upstream primer 10 μM 2 Downstream primer 10 μM 2 Fluorescent probe primer 5 μM 2 DNA template 5 wxya 2 o

22

[0050] Table 3 The reaction conditions of the African swine fever virus multiplex real-time fluorescent PCR method

[0051]

Embodiment 2

[0052] The optimization of embodiment 2 African swine fever virus real-time fluorescent PCR detection method

[0053] 1. Reaction system of African swine fever virus real-time fluorescent PCR method

[0054] The optimization principle of the real-time fluorescent PCR method for African swine fever virus is: by optimizing the following conditions, the same sample can obtain the maximum amplification efficiency and the minimum Ct value.

[0055] Commercial 5U / μl Taq enzyme is generally added 2μl to the 50μl reaction system, and the final concentration is 0.2U / μl. In this example, under the condition of further reducing the final concentration of Taq enzyme, the reagent The original chemical reagent components of the kit were combined into two types, and a specific enzyme mixture and PCR reaction solution were prepared to further optimize the detection conditions of the kit, greatly improving the sensitivity and specificity. After optimization, the reaction system of the real-ti...

Embodiment 3

[0071] Example 3 PCR detection kit sensitivity and specificity comparison test

[0072] With African swine fever virus p72 gene shown in embodiment 1, CD2v gene, MGF360-14L gene primer pair, probe combination, according to embodiment 1 condition optimization front component assembly African swine fever virus triple real-time fluorescent PCR kit 1, implement Example 2: African swine fever virus triple real-time fluorescent PCR kit 2 assembled from components after condition optimization. The sensitivity and specificity of the two kits were compared.

[0073] After the African swine fever virus DNA template was serially diluted 10 times, kit 1 and kit 2 were subjected to PCR sensitivity tests according to the pre-optimization and post-optimization conditions, respectively.

[0074]The results showed that the sensitivity of kit 1 was as low as 10 copies, and the sensitivity of kit 2 was as low as 4 copies. It shows that the primers screened by the present invention have high se...

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Abstract

The invention relates to a real-time fluorescent PCR amplification primer group for identifying and testing African swine fever viruses, the amplification primer group consists of upstream and downstream primers and probe primers of an African swine fever virus p72 gene, a CD2v gene and an MGF360-14L gene, and the invention also relates to a kit containing the real-time fluorescent PCR amplification primer group for identifying and testing the African swine fever viruses. By utilizing the high sensitivity of the primer group, wild strain infection or gene deletion vaccine strain immunization can be distinguished, the African swine fever virus infection can be prevented and controlled as soon as possible, and meanwhile, the kit can accurately detect suspected infection samples from different regional sources.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a pair of real-time fluorescent PCR amplification primers and probe primers for identifying and testing African swine fever virus, and a kit containing the pair of real-time fluorescent PCR amplification primers and probe primers. Background technique [0002] African swine fever (African swine fever, ASF) is a severe, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV). African swine fever virus is an enveloped single-molecule linear double-stranded DNA virus with a genome length of about 170kb to 193kb. ASF has the characteristics of high morbidity and mortality. Once it occurs, it will cause huge economic losses. At present, there is a lack of effective vaccines and specific treatment methods, making ASF one of the most serious diseases that endanger the pig industry. At present, the control of ASF can only rely on the rapid diagn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2563/107C12Q2561/113C12Q2545/113
Inventor 田克恭李向东张海洋
Owner LUOYANG PULIKE WANTAI BIOTECH
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