Molecular probe for detecting African swine fever virus, kit and application thereof

An African swine fever virus and detection kit technology, applied in biological testing, measuring devices, analytical materials, etc., can solve the problems of poor reliability and practicability of detection technology, and achieve advantages of application and promotion, high sensitivity, and repeatability. good effect

Active Publication Date: 2019-05-21
河南中泽生物工程有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a molecular probe for detecting African swine fever virus (ASFV), a kit and its application, so as to solve the technical problem of poor reliability and practicability of the existing detection technology

Method used

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  • Molecular probe for detecting African swine fever virus, kit and application thereof
  • Molecular probe for detecting African swine fever virus, kit and application thereof
  • Molecular probe for detecting African swine fever virus, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: Design a kind of molecular probe for detecting ASFV

[0061] According to the 54 strains of African swine fever virus VP72 genes on GeneBank, a conserved sequence with a nucleotide sequence size of 258 bp at the 3' end of the VP72 gene was screened out, as shown in SEQ ID NO.1:

[0062] CGTATCCGATCACATTACCTATTATTAAAAACATTTCCGTAACTGCTCATGGTATCAATCTTATCGATAAATTTCCATCAAAGTTCTGCAGCTCTACATACCCTTCCACTACGGAGGCAATGCGATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGTCATATTAACGTATCCAGAGCAAGATTGAATG.

[0063] Taking the conserved sequence as the target gene, considering various factors and combining years of practical experience, a molecular probe was designed, as shown in SEQ ID NO.2:

[0064] 5'-[6-carboxy-fluorescein (FAM)]-CCAACCCAGTGGTCATATTAACGTATCC-3'-[6-carboxy-tetramethyl-rhodamine (TAM-RA)].

[0065] The fluorescent reporter group labeled at the 5' end of the probe is 6-carboxy-fluorescein (FAM), and the fluorescent quencher grou...

Embodiment 2

[0066] Embodiment 2: design a kind of specific primer for detecting ASFV

[0067] Taking the conserved sequence shown in SEQ ID NO.1 as the target gene, a pair of specific primers were designed by comprehensive consideration of various factors and years of practical experience, as shown in SEQ ID NO.3 and SEQ ID NO.4:

[0068] ASFV-For: 5'-CGTATCCGATCACATTACC-3'

[0069] ASFV-Back: 5'-CGTAATCCGTGTCCCAAC-3'

Embodiment 3

[0070] Embodiment 3: extract ASFV total DNA

[0071] (1) Reagent preparation:

[0072] Separation buffer: 10mmoL / L Tris-Cl pH 7.4, 10mmoL / L NaCl, 25mmoL / L EDTA;

[0073] Other reagents: 10% SDS, protein kinase K (20mg / mL or powder), diethyl ether, phenol: chloroform: isoamyl alcohol (25:24:1), absolute ethanol and 70% ethanol, 5 mol / L NaCl, 3 mol / L NaAc, TE;

[0074] (2) Cut off about 5g of tissue, remove the connective tissue, absorb the blood with absorbent paper, cut it into pieces (the more broken the better), and put it in a mortar;

[0075] (3) Pour into liquid nitrogen, grind into powder, add 10mL separation buffer;

[0076] (4) Add 10 mL 10% SDS, mix well, and the sample becomes very viscous at this time;

[0077] (5) Add 50 μL or 1 mg of protein kinase K and incubate at 37 °C for 1-2 h to guide the complete dissociation of the tissue;

[0078] (6) Add 1 mL 5mol / L NaCl, mix well, and centrifuge at 5000 rpm for a few seconds;

[0079] (7) Take the supernatant in a...

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Abstract

The invention discloses a molecular probe for detecting African swine fever virus, a kit and application thereof, so as to solve the technical problems of poor reliability and poor practicability of the existing detection technology. The nucleotide sequence of the molecular probe for detecting African swine fever virus is shown as in SEQ ID NO.2. An RT-PCR reaction solution for detecting African swine fever virus is prepared, lyophilized powder for the RT-PCR reaction solution for detecting African swine fever virus is prepared, an African swine fever virus detection kit is prepared, and an African swine fever virus qualitative and quantitative detection method is provided. The designed molecular probe and the built RT-PCR detection method have the characteristics of high sensitivity, goodrepeatability and strong specificity, the reliability and the usability of ASFV detection by the PCR technology are improved, pollution-free and high-flux detection is realized, and ASFV nucleic acids in susceptible animals and related animal products can be detected.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a molecular probe for detecting African swine fever virus, a kit and an application thereof. Background technique [0002] African swine fever virus (ASFV) infecting domestic animals or wild boars causes African swine fever (ASF), which can cause high morbidity and mortality and cause huge economic losses to the swine industry. ASFV was first discovered in Kenya in 1921, and then ASFV was found in many countries in southern and eastern Africa. ASF spreads rapidly and has a rapid onset. It has spread from Africa to all parts of the world in just a few decades, and has almost swept the world. At 17:00 on August 2, 2018, China Center for Animal Health and Epidemiology diagnosed a suspected African swine fever epidemic in Wuwu Community, Shenbei Street (Xinchengzi), Shenbei New District, Shenyang City. Diagnosed at 11 o'clock. As of 15:00 on August 3, 2018, all 913 pigs in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58G01N21/64
Inventor 王爱萍张改平刘运超贾蕊刘东民赵建国祁元明石海宁
Owner 河南中泽生物工程有限公司
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