African swine fever virus wild strain and vaccine strain identification and detection kit

An African swine fever virus and wild virus strain technology is applied in the field of identification and detection kits for African swine fever virus wild virus strains and vaccine strains. high effect

Inactive Publication Date: 2020-11-03
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study found that the deletion of part of the MGF360 / 505 gene or the EP402R gene (encoding CD2v protein) of the multi-gene family member can reduce the virulence of African swine fever virus

Method used

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  • African swine fever virus wild strain and vaccine strain identification and detection kit
  • African swine fever virus wild strain and vaccine strain identification and detection kit
  • African swine fever virus wild strain and vaccine strain identification and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Preparation and Application of Triple Fluorescent PCR Identification Detection Kit for African Swine Fever Virus Wild Strain and Vaccine Strain

[0087] 1. Preparation of triple fluorescent PCR identification kit for African swine fever virus wild strain and vaccine strain

[0088] 1. Synthesis of Primers and Probes

[0089] The following primers and probes were artificially synthesized:

[0090] Primer P72F3: 5'-GTTTCGGTACGCATTCTTTGTG-3';

[0091] Primer P72R3: 5'-CGGATATGACTGGGACAACCA-3';

[0092] Probe P72P3: 5'-Cy5-ATCTACAAGCGTGTAAACGGCGCCC-BHQ-3-3'

[0093] The 5'-end of the probe P72P3 is labeled with the fluorescent reporter group Cy5, and the 3'-end is labeled with the fluorescent quencher group BHQ-3.

[0094] CD2vF2: 5'-CCCAATATCCCGCCATTATCT-3'

[0095] CD2vR2: 5'-AAATGAAGACCAGCTTGAAAGGTT-3'

[0096] CD2vP2: 5'-VIC-TTCGCTTATTCACGTAGATAG-BHQ1-3'

[0097] The 5'-end of the probe CD2vP2 is labeled with the fluorescent reporter group VIC, and the...

Embodiment 2

[0118] Example 2. Detection of Sensitivity of African Swine Fever Virus Field Strain and Vaccine Strain Triple Fluorescence PCR Differentiation Detection Kit

[0119] The sensitivity test was performed using the kit prepared in Example 1.

[0120] 1. Preparation of template DNA

[0121] With the step 1 of embodiment 1, prepare final concentration respectively and be 3 * 10 6 Copy / μL B646L gene, EP402R gene and mCherry gene positive standard, take 100 μL each into a 1.5mL centrifuge tube, and obtain a final concentration of 1×10 6 Copies / μL of B646L gene, EP402R gene and mCherry gene positive standard mixture. Carry out 10-fold serial dilutions to the positive standard mixture with double distilled water, prepare dilution I respectively (concentration is 1×10 6 copy / μL), diluent II (each concentration is 1×10 5 copy / μL), diluent III (concentration is 1×10 4 copy / μL), diluent IV (concentration is 1×10 3 copy / μL), diluent V (each concentration is 1×10 2 copies / μL) and dilu...

Embodiment 3

[0135] Example 3. Detection of specificity of African swine fever virus wild strain and vaccine strain identification kit

[0136] The kit prepared in Example 1 was used for specific detection.

[0137] 1. Sample processing

[0138] Take 200 μL each of the virus solutions of African swine fever virus LNSY wild strain, ΔCD2vΔMGF vaccine candidate strain, CSF virus vaccine strain, and foot-and-mouth disease virus vaccine strain, and use a commercial virus nucleic acid extraction kit (Qiagen) to extract nucleic acid.

[0139] 2. Fluorescent PCR detection

[0140] Prepare the reaction master mix. The 25 μL reaction system contains 12.5 μL of 2×PCR reaction buffer and 10.5 μL of double distilled water.

[0141] WT tube: In a 0.2mL centrifuge tube, add 2μL of African swine fever virus LNSY wild strain nucleic acid, and then add 23μL of premix.

[0142] Vac tube: In a 0.2mL centrifuge tube, add 2μL of African swine fever virus ΔCD2vΔMGF vaccine candidate strain nucleic acid, and the...

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Abstract

The invention discloses an African swine fever virus wild strain and vaccine strain identification and detection kit and application thereof. The kit provided by the invention comprises three groups of primers and probes, and the first group of primers and probes is completely paired with a specific sequence of an African swine fever virus B646L gene conserved region and is used for detecting theAfrican swine fever virus; the second group of primers and probes is completely paired with a specific sequence of an African swine fever virus EP402R gene conserved region and is used for detecting the African swine fever virus wild strain; and the third group of primers and probes is completely paired with the specific sequence of a red fluorescent protein mCherry gene and is used for detectingthe African swine fever virus vaccine strain. Different fluorescent reporter groups are respectively marked on the three probes. The African swine fever virus wild strain and vaccine strain identification and detection kit is high in detection sensitivity, capable of detecting target DNA with the minimum concentration of 100 copies/[mu] L, good in specificity, easy and convenient to operate and capable of effectively distinguishing African swine fever vaccine immune animals from wild virus infected animals.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a detection kit for distinguishing African swine fever virus wild strains and vaccine strains. Background technique [0002] African swine fever (ASF) is an acute, severe and highly contagious disease of pigs caused by African swine fever virus (ASFV) infection. Pigs of different breeds and ages are susceptible, with a high incidence rate and a lethal rate of up to 100%. There is currently no effective treatment and no vaccine available for the disease. African swine fever was first discovered in Kenya, Africa in 1921. It was introduced to Portugal in 1957, and then spread to other European countries. By 1995, African swine fever was eradicated in Europe except in southern Sardinia. African swine fever spread from Spain to South America in 1971 and was eradicated in the region in 1984. In 2007, African swine fever was introduced into Georgia and spread in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2563/107
Inventor 包静月王清华戈胜强王淑娟张永强吕艳
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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