Dual fluorescent PCR primer and kit for identifying ASFV strain and CD2v gene-deleted strain

A gene deletion and dual fluorescence technology, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems that wild strains are difficult to identify and cannot be identified

Inactive Publication Date: 2019-12-31
青岛立见生物科技有限公司
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Problems solved by technology

But at the same time, the gene deletion strain also has the problem of being difficult to distinguish from the wild strain
In addition to the difference in pathogenicity, other characteristics of the gene deletion strain are very similar to those of the wild strain. They can also grow and proliferate in pigs for a period of time, and they cannot be identified by the existing fluorescent PCR method.

Method used

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  • Dual fluorescent PCR primer and kit for identifying ASFV strain and CD2v gene-deleted strain

Examples

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Effect test

Embodiment 1

[0028] 1. Design and synthesis of specific primers and probes for African swine fever virus p72 gene and CD2v gene According to the complete genome sequence of African swine fever virus (MK333180) published on GenBank, use Primer Primer5.0 software to design and detect p72 gene respectively and CD2v gene-specific primers and probes, the sequences are as follows:

[0029] SEQ ID NO.1: 5'-FAM-TATCGATAAGATTGAT-MGB-3';

[0030] SEQ ID NO.2: 5'-HEX-CACCACTTCCATACATGAACCATCTCCCCA-BHQ1-3';

[0031] SEQ ID NO.3: 5'-ATAGAGATACAGCTCTCTTCCAG-3';

[0032] SEQ ID NO.4: 5'-GTATGTAAGAGCTGCAGAAC-3';

[0033] SEQ ID NO.5: 5'-GAAGAAGAACAATGTCAGCATGAT-3';

[0034] SEQ ID NO.6: 5'-AACGACTGTAAGGCTTAGGAAGTAATG-3';

[0035] Wherein, the 5' end and 3' end of SEQ ID NO.1 are marked with FAM and MGB respectively, and a set of primers for detecting p72 gene is formed with SEQ ID NO.3 and SEQ ID NO.4;

[0036] The 5' end and 3' end of SEQ ID NO.2 are marked with HEX and BHQ1 respectively, and a set ...

Embodiment 2

[0059] Detection of African swine fever virus in pig serum after pig farm immunization African swine fever vaccine of embodiment 2

[0060] 1.1 Source of samples

[0061] There are 2,000 sows in a pig farm in a certain place. They have been immunized with a certain African swine fever vaccine that is in the pilot stage for one month. Some pigs still die sporadically. Now some serum is collected for detection of African swine fever virus.

[0062] 1.2 Processing of samples

[0063] Use a DNA extraction kit or an automatic nucleic acid extractor to extract the DNA in the collected serum samples, and store them at low temperature for testing.

[0064] 1.3 Amplification reagent preparation

[0065] Calculate the amount of solution according to the number of test samples. Each PCR reaction contains 12.5 μl of fluorescent PCR reaction solution and 7.5 μl of primer mixture. Take 5 μl of negative control, 5 μl of DNA to be tested and 5 μl of positive control (mix well) and add them...

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Abstract

The invention relates to double fluorescent PCR primers and a kit for identifying ASFV strain and CD2v gene-deleted strains, and belongs to the technical field of biology. The sequences of the primersare shown as SEQ ID NO.1-6. The invention also discloses the kit comprising the primers. The kit is composed of the following components: a fluorescent PCR reaction solution, a mixed solution containing the primers, a positive control and a negative control. The six primers are all placed in one tube, are mixed together for use, do not interfere with one another, do not influence subsequent PCR amplification and fluorescence signal value acquisition, and can identify African swine fever virus wild strain and gene-deleted strains.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for identifying and detecting African swine fever virus wild strains and gene deletion strains. Background technique [0002] African swine fever virus (ASFV) is the only member of the African swine fever virus family. Its genome is a single-stranded DNA virus with a size of 150-190kb and can encode at least more than 150 proteins. ASFV can infect domestic pigs and wild boars at various stages, and the mortality rate is as high as 100%. ASFV can be transmitted through direct and indirect contact, and ASFV-contaminated pork, cured ham, swill, feed, means of transportation, and personnel can all become sources of infection. Since the African swine fever epidemic was first discovered in 1921, the African swine fever virus has spread from Africa to Europe, Asia and other places through various means such as airplanes and ships. In 2018, African swine fever also broke ou...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 张志李翠翠孙静张善鹏宫枫举官丽娟孙学强
Owner 青岛立见生物科技有限公司
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