Quick identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type

A base and fluorophore technology, which is applied in the field of detection of African swine fever virus genotype I and II specific difference sites and rapid differential diagnosis, can solve the problem of no effective treatment methods

Active Publication Date: 2019-11-12
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the specific immune evasion mechanism of th

Method used

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  • Quick identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type
  • Quick identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type
  • Quick identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type

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Experimental program
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Effect test

Embodiment 1

[0047] In order to further explore the impact of sample concentration on the specificity of the RAA method, the present invention constructed ASFV gene type I and type II plasmids, and carried out a series of dilutions respectively, including 10 5 ,10 4 ,10 3 ,10 2 , 10 1 The copies / reactions were then detected separately using the RAA method (A and G reactions) (Fig. 3).

[0048] in Figure 3-1 It is the amplification situation of the ASFV type I plasmid of different concentrations in A reaction, and the concentration is 10 2 -10 5 The amplification gradient of the ASFV type I plasmid in A reaction is obvious; Figure 3-2 It is the amplification situation of the ASFV type I plasmid of different concentrations in the G reaction, and the concentration is 10 1 -10 5 The amplification gradient of the ASFV type I plasmid in the G reaction is not obvious, only 10 4 -10 5 The amplification line is more obvious, and than Figure 3-1 Middle A reaction amplification line com...

Embodiment 2

[0050] In order to explore the specificity of A reaction and G reaction, African swine fever (ASFV) I and II genes, foot-and-mouth disease (FMD), porcine parvovirus (PPV), pseudorabies virus (PRV), porcine circovirus (PCV) , Japanese encephalitis (JEV), blue ear disease (PRRV), swine fever virus (CSFV), transmissible gastroenteritis virus (TGEV) nucleic acid as a template, RAA method (A and G reaction) detection ( Figure 4).

[0051] in Pic 4-1 For the specific detection of A reaction, only the ASFV genotype I has obvious amplification lines, indicating that the A reaction has good specificity for detecting ASFV genotype I; Figure 4-2 For the specific detection of G reaction, only the ASFV genotype II has obvious amplification lines, indicating that the G reaction has good specificity for detecting ASFV genotype II.

Embodiment 3

[0053] 1. Collection of clinical specimens

[0054] DNA from porcine tissue and whole blood samples was extracted by Tianlong Automatic Nucleic Acid Extractor, and stored at -20°C for later use. The DNA concentration of the obtained samples ranged from 10 to 75 ng / μL.

[0055] 2. RAA detection was performed on 20 clinical samples

[0056] The RAA fluorescence method basic reaction unit kit produced by Jiangsu Qitian Gene Company was used. Both reactions A and C were carried out in a 0.2ml reaction tube, which contained pre-lyophilized enzyme mixture (SSB, UvsX, DNA polymerase, Exonuclease III), then added 25μL reaction buffer solution, 17μl nuclease-free water , 2.1 μL forward primer (15 μM), 1.4 μL corresponding specific reverse probe (15 μM), 2 μL template (10-75 ng / μL), 2.5 μL magnesium acetate (280 mM). After the solution was prepared, it was transferred to the QT-RAA-F7200 fluorescence detector produced by Jiangsu Qitian Company for detection, and the reaction was carr...

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Abstract

The invention provides an identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type based on a probe-oriented recombinase-mediated isothermal amplification technique. The method can be used for quickly diagnosing and distinguishing the African swine fever virus gene I type and the African swine fever virus gene II type. The invention relates to a probe for detecting specific difference sites of different genotypes based on the probe-oriented recombinase-mediated isothermal amplification method. According to the probe, based on the probe used in a recombinase-mediated isothermal amplification method, tetrahydrofuran is used for replacing a backward base of a site or a backward base of a specific difference site complementary site,excision enzymes III are subjected to enzymolysis until cutting of a tetrahydrofuran site is finished, a cutoff fluorophore modified base produces fluorescence signals in a system, and remaining probe parts exert the effect of a downstream primer. The probe can simplify necessary downstream primer components in the detection course, and a downstream primer and the probe are combined for use, so that the possibility that the probe and the primer generate non-specific amplification is reduced.

Description

technical field [0001] The invention belongs to the technical field of poultry disease detection, and in particular relates to a rapid differential diagnosis method for African swine fever virus genotype I and II, that is, a probe-guided recombinase-mediated isothermal amplification technology is applied to the detection of African pigs Pestivirus genotype I and II specific differential loci and rapid differential diagnosis method. Background technique [0002] African swine fever (ASF) is an acute, contagious infectious disease of pigs caused by African swine fever virus (ASFV). The World Organization for Animal Health (OIE) listed it as a Class A infectious disease. Due to the special immune evasion mechanism of the virus, there is currently no effective treatment. Therefore, the detection and quarantine of the virus is particularly important. [0003] African swine fever virus has only one serotype, but the existing circulating strains can be divided into 24 genotypes ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2525/117C12Q2521/319C12Q2563/107
Inventor 樊晓旭吴晓东赵洋蔡禹希王清华包静月赵明胡永新戈胜强李林刘春菊应清界
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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