PCR primer group and kit for detecting African swine fever viruses based on double genes and application

An African swine fever virus and detection kit technology, applied in the field of PCR primer sets, can solve the problems of cumbersome operation, waste of detection reagents, and reduced detection sensitivity, and achieve the effect of ensuring specificity and sensitivity

Active Publication Date: 2020-06-02
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In CN110373500A, a kind of test kit and application thereof based on double fluorescent PCR detection of B646L (p72) and B438L (p49) double genes are mentioned, but its defect is that, compared with single-gene detection, this scheme will be to a certain extent Reduced sensitivity of detection
In addition, the positive control in this technical solution is the B646L (p72) and B438L gene tandem plasmid vector, which cannot effectively

Method used

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  • PCR primer group and kit for detecting African swine fever viruses based on double genes and application
  • PCR primer group and kit for detecting African swine fever viruses based on double genes and application
  • PCR primer group and kit for detecting African swine fever viruses based on double genes and application

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0071] Example 1 Design of specific primers and probes for double-gene detection of African swine fever virus

[0072] The present invention downloads B64L6 (p72) and E183L (p54) gene sequences of different ASFV strains from a database for comparison and analysis, and designs primers in the front, middle and back segments of the gene according to the analysis results for screening. In order to improve the specificity and sensitivity of detection and realize high-efficiency double-gene real-time fluorescent quantitative PCR detection, the present invention aims at the affinity of primers, probes and target sequences, the secondary structure between primers, and the secondary structure between primers and target sequences. The structure, GC content of primers, Tm value, length and length of amplified fragments have been designed and screened manually. Finally, the best primer and probe combination as shown in SEQ ID NO.1-6 was obtained.

[0073] The following is a partial listing of...

Example Embodiment

[0092] Example 2 The optimal primer sequence of p72 is combined with p54, E184L, K196R, and B438L

[0093] In this example, the double gene detection combination of p72+p54, p72+E184L, p72+K196R, p72+B438L was performed respectively, and the mixture of artificially synthesized p72, p54, E184L, K196R, B438L gene plasmids, E184L, K196R, B438L Primers and probe sequences are derived from references and patent applications (see Table 6 below); take a fluorescent quantitative PCR reaction tube, add 2×PCR reaction solution 10μL, B646L(p72) gene primer probe mixture 1.5μL, E183L( p54), E184L, K196R, B438L gene primer probe mixture was added 1.5μL, 7μL of the sample DNA to be tested, and the reaction system of fluorescence quantitative PCR was performed; the primer probe mixture contained 10μM primer mixture (SEQ ID NO. 1-4) and 5μM probe mixture (SEQ ID NO.5-6); the PCR reaction solution contains DNA polymerase, reaction buffer (100mM KCl, 20mM Tris-HCl (pH 8.5), 4mMMgCl2), dNTP Mixtur...

Example Embodiment

[0102] Embodiment 3 ASFV p72, p54 double gene detection kit

[0103] This embodiment provides a kit for ASFV dual-gene real-time fluorescent quantitative PCR detection. The kit contains the following components: 2×PCR reaction solution (including DNA polymerase, reaction buffer (100mM KCl, 15mM Tris-HCl (pH 8.5), 4mM MgCl2), dNTP mixture (0.5mM each)), p72 primer probe mixture (primers p72-F and p72-R concentration are both 10μM, probe p72-P concentration is 5μM), p54 primer probe Needle mixture (the concentration of primers p54-F and p54-R are both 10μM, and the concentration of probe p54-P is 5μM), reference substance.

[0104] Among them, the 2×PCR reaction solution is a commercial product of Bao Biological Engineering (Dalian) Co., Ltd. (product code RR390).

[0105] The reference substance is an artificially designed synthetic plasmid containing DNA fragments composed of upstream primers for detecting African swine fever virus B64L6 (P72) gene, probe primers and downstream prim...

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Abstract

The invention relates to the technical field of virus detection, in particular to a PCR primer group and kit for detecting African swine fever viruses based on double genes and application. The primergroup and method are high in detection specificity, good in repeatability and high in sensitivity, and meanwhile have the advantages of being easy and convenient to operate, short in detection time and the like, false positive results can be effectively reduced, the influence of positive control on a detection environment is reduced, and the primer group and the kit have high application value inrapid detection of the African swine fever viruses.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a PCR primer set, kit and application for detecting African swine fever virus based on double genes. Background technique [0002] African swine fever (ASF) is a severe infectious disease caused by African swine fever virus (ASFV). animal disease. ASF does not infect humans, and the fatality rate after infecting pigs can reach 100%. Clinical symptoms and pathology of pigs infected with ASFV. [0003] The changes are very similar to classical swine fever, and it is easy to confuse the two diseases. The main manifestations are high fever (the body temperature of pigs is generally as high as 40.5-42°C), cyanosis of the skin with bleeding spots, and even a large number of asymptomatic dead pigs, among which abnormal enlargement of the spleen is the typical symptom. Pigs are the only domestic animals naturally infected with ASFV, and wild boars are also susceptible. The cl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 王金良沈志强陈金龙于新友董林胡绍良
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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