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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 reduce the probability of false positives in the detection method if used for a long time; at the same time, the sample adding system involved in the invention is cumbersome to operate. A volume of 0.1 μL needs to be added to the single-well system. The sample volume of the template is 1 uL, and the sample is added 6 times. There are certain requirements for the operator, and the detection reagent will also cause unnecessary waste.

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

Embodiment 1

[0071] Embodiment 1 is used for the design of the specific primer of African swine fever virus double gene detection and probe

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

Embodiment 2

[0092] Example 2 The optimal primer sequence for p72 and p54, E184L, K196R, B438L for double gene combination

[0093] In this example, p72+p54, p72+E184L, p72+K196R, p72+B438L double-gene detection combination, fluorescent detection of artificially synthesized p72, p54, E184L, K196R, B438L gene plasmid mixture, E184L, K196R, B438L The sequences of primers and probes are derived from references and patent applications (see Table 6 below); take a fluorescent quantitative PCR reaction tube, add 10 μL of 2×PCR reaction solution, 1.5 μL of B646L (p72) gene primer-probe mixture, and E183L ( p54), E184L, K196R, and B438L gene primer-probe mixtures were added to 1.5 μL respectively, and 7 μL of sample DNA to be tested was used to carry out a reaction system for fluorescent quantitative PCR; the primer-probe mixtures contained 10 μM primer mixtures (SEQ ID NO. 1-4) and 5 μ M probe mixed solution (SEQ ID NO.5-6); The PCR reaction solution comprises DNA polymerase, reaction buffer (100m...

Embodiment 3

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

[0103] The present embodiment provides the test kit that is used for ASFV double-gene real-time fluorescent quantitative PCR detection, and this test kit comprises following components: 2 * PCR reaction solution (comprising DNA polymerase, reaction buffer (100mM KCl, 15mM Tris-HCl (pH 8.5), 4mM MgCl2), dNTP mixture (each 0.5mM)), p72 primer probe mixture (concentration of primer p72-F and p72-R is 10μM, probe p72-P concentration is 5μM), p54 primer probe Needle mixture (the concentrations of primers p54-F and p54-R are both 10 μM, and the concentration of probe p54-P is 5 μM), and reference substances.

[0104] Among them, the 2×PCR reaction solution is a commercially available product from Treasure Bioengineering (Dalian) Co., Ltd. (product code RR390).

[0105] The reference substance is an artificially designed synthetic plasmid, which contains a DNA fragment composed of upstream primers for detecting the African s...

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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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