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Primer group and kit for African swine fever virus LAMP detection and application

A technology of African swine fever virus and primer set, applied in the direction of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problems of easy false positives, large impact on results, high cost, etc., and avoid aerosol pollution, method Intuitive and convenient, low equipment requirements

Inactive Publication Date: 2019-04-16
许昌佰柯蛋白与基因工程研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, virus isolation and identification, in vitro amplification and culture time is long, cell culture requires high personnel and equipment, and the steps are cumbersome; immunohistochemistry, the detection of viruses in diseased tissue, has poor specificity, false positives are prone to occur, and the operation process is complicated. Preservation and processing of disease materials require professionals to operate, and the differences in the operation process have a great impact on the results, and the tester needs to have certain experience to make an accurate judgment; enzyme-linked immunosorbent adsorption, the result is more accurate, but the sample requirements are relatively high. High, fresh disease material is required to detect accurately, detection requires antibodies, high cost, long time, at least 4 to 6 hours to get results; reverse transcription polymerase chain reaction requires professional equipment such as PCR machine, which needs to be carried out in the laboratory operate

Method used

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  • Primer group and kit for African swine fever virus LAMP detection and application
  • Primer group and kit for African swine fever virus LAMP detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 A kind of method utilizing LAMP technology to detect African swine fever virus

[0048] The detection process is strictly partitioned

[0049] Area 1: Reagent preparation area----prepare the required reagents

[0050] Area 2: Sample preparation area - processing of samples to be tested and control samples

[0051] Three areas: reaction area ----- amplification and result analysis

[0052] 1. Experimental materials

[0053] Supernatant of organ grinding fluid from diseased pigs infected with African swine fever virus

[0054] 2. Genomic DNA extraction (in the second area)

[0055] 1) Take 200 μL of organ grinding supernatant, add an equal amount of lysis buffer A to 750 μL of whole blood, mix well, centrifuge at 12000 r / min for 30 s, and discard the supernatant.

[0056] 2) Suspend the pellet with 1.5mL lysis buffer A, repeat centrifugation once, and discard the supernatant.

[0057] 3) Dissolve the precipitate with 117 μL of lysis buffer B, act at 70°...

Embodiment 2

[0084] The specific detection of the LAMP detection method of embodiment 2 African swine fever virus

[0085] African swine fever virus samples, porcine blue ear virus, porcine parvovirus samples, and healthy pig samples were detected using the kit in Example 1 of the present invention.

[0086] 1) Sampling pig samples infected with African swine fever virus, blue ear virus and porcine parvovirus, taking 0.1g tissue, adding an equal amount of lysis buffer A to 750μL whole blood, mixing, centrifuging at 12000r / min for 30s, discarding supernatant.

[0087] 2) Suspend the pellet with 1.5mL lysis buffer A, repeat centrifugation once, and discard the supernatant.

[0088] 3) Dissolve the precipitate with 117 μL of lysis buffer B, act at 70°C for 5 minutes, add 3 μL of proteinase K when cooled to 55°C, and incubate for 1 hour to digest the cells, and inactivate proteinase K at 95°C for 10 minutes.

[0089] 4) Add 30 μL of 2 mol / L KCL solution and melt on ice for 5 min.

[0090] 5...

Embodiment 3

[0096] The sensitivity detection of the LAMP detection method of embodiment 3 African swine fever virus

[0097] The PA104R gene of African swine fever virus was artificially synthesized by Shanghai Sangon Bioengineering Co., Ltd., connected to the vector PMD18T, transformed into Escherichia coli DH5a, picked a single clone into the liquid medium, and extracted the plasmid with a plasmid extraction kit. Use a nucleic acid and protein analyzer to measure the OD260 value, and convert the plasmid copy number according to the formula.

[0098] (6.02×10 23) ×(ng / μL×10 -9 ) / (DNAlength×660)=copies / μL.

[0099] The plasmid copy number was determined to be 5x10 6 copies / μL, carry out 10-fold serial dilution of the plasmid, the concentration is 5x10 5 , 5x10 4 , 5x10 3 , 5x10 2 , 5x10 1 , 5copies / μL, using the LAMP detection method of Example 1, the same reaction conditions and reaction system, carry out the LAMP sensitivity test and the PCR sensitivity test, wherein the LAMP me...

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Abstract

The invention provides a primer group and kit for African swine fever virus LAMP detection and application, and belongs to the technical field of biological detection. The kit has the advantages thatthe operation is simple, the requirements on equipment is low, and amplification can be completed by putting a detected sample and detection reaction liquid into a constant-temperature water bathing pot with the temperature of 58 DEG C to be hatched for 50 min when detection is conducted, the flexibility and specificity of the kit are high; the result is judged by observing the color changing of the reaction liquid through naked eyes, if the reaction liquid is yellow, it is indicated that African swine fever virus positive is detected, and if the reaction liquid is orange, it is indicated thatAfrican swine fever virus negative is detected. The method has the advantages of being visual and direct, and the aerosol contamination caused by the opening of a cover after the reaction is conducted can be effectively avoided.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of viruses, in particular to a primer set, kit and application for detection of African swine fever virus LAMP. Background technique [0002] African swine fever (ASF), caused by African swine fever virus (ASFV), is a porcine viral infectious disease that seriously threatens the pig industry. The onset of ASF has atypical, acute, subacute and chronic forms. Mortality varies from 0% to 100% due to the different virulence of the viruses infected with pigs. Acute disease is characterized by a short incubation period, about 3 to 7 days, accompanied by high fever (up to 42°C), and death within 5 to 10 days. [0003] African swine fever virus belongs to DNA virus order, African swine fever virus family, member of African swine fever virus genus. It is a icosahedral structure with a diameter of 175-215nm, a genome length of 170-190kb, and contains 151 open reading frames. It can en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 王学庆李鹏云王楠
Owner 许昌佰柯蛋白与基因工程研究院有限公司
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