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Crossing isothermal amplification primer set for detecting pseudorabies virus wild strain, kit and application thereof

A pseudorabies virus and isothermal amplification technology, which is applied in the field of warm amplification primer sets, can solve problems that hinder application and promotion, and achieve intuitive and accurate reaction results, good specificity, and rapid interpretation

Pending Publication Date: 2017-05-31
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology requires expensive thermal cyclers and skilled operators during the amplification process, which hinders the wide application and promotion of this technology in veterinary grassroots

Method used

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  • Crossing isothermal amplification primer set for detecting pseudorabies virus wild strain, kit and application thereof
  • Crossing isothermal amplification primer set for detecting pseudorabies virus wild strain, kit and application thereof
  • Crossing isothermal amplification primer set for detecting pseudorabies virus wild strain, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Design and synthesis of embodiment 1 cross primer constant temperature amplification primer

[0047] According to the principle of primer design, for the conserved region sequence of the gE gene of the wild strain of pseudorabies virus, use Priemer5 software to design primers. According to our experience, the GC content of the primers is guaranteed to be between 40% and 60%, and the Tm value of each primer is 50%. ~60°C or so. Then we used the PrimerSelect tool of Larsergene7.0 biological software to preliminarily screen the primers. The screening principle was to ensure that the dG value between each primer pair was small. The primer sequences are shown in Table 1 (B2 and B1 primers are not biomarked at this time), the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the synthesized primers were diluted with sterilized three-distilled water to a concentration of 10 μM, - Store at 20°C.

Embodiment 2

[0048] Example 2 Application of Cross Primer Constant Temperature Amplification Primer in Detecting the Virus to be Tested

[0049] 1. Extraction of viral nucleic acid

[0050] Take 200 μL of the sample, and extract the nucleic acid of the virus according to the operating instructions of the viral RNA / DNA extraction kit.

[0051] 2. Establishment of cross isothermal amplification method

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Abstract

The invention discloses a crossing isothermal amplification primer set for detecting a pseudorabies virus wild strain, a kit and application thereof. The primer set comprises the primer set with the nucleotide sequences shown as SEQ ID NO.1-5. The kit comprises the primer set and a nucleic acid test strip. The use method of the kit comprises the following steps: firstly, preparing a crossing primer amplified reaction system, detecting a product acquired through thermostatic reaction with the nucleic acid test strip and then directly reading: wherein two strips indicate a positive result, one strip is located in a detection area and the other strip is located in a quality control area. The kit has the advantages of simple operation, low cost, easily observed reaction result, excellent specificity, suitability for on-site detection for export quarantine, food hygiene and livestock farm and easiness in wide application and popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cross isothermal amplification primer set, kit and application for detecting pseudorabies virus wild strains. Background technique [0002] Porcine pseudorabies (Pseudorabies, PR) is caused by pseudorabies virus (Pseudorabies Virus, PRV) infection, and is an important infectious disease that seriously endangers the pig industry in my country. Pseudorabies virus belongs to the αherpesvirus family. The genome is a double-stranded DNA molecule with a size of about 150kb. The mature virion carries more than 50 kinds of proteins, among which virulence genes such as gB, gE, gG, gI, and TK are non-essential for its replication. Although my country once adopted the strategy of combining gE gene deletion vaccine immunization with wild virus infection antibody monitoring to achieve the control and purification of the disease, the emergence of mutant strains in pigs at the end of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6848C12Q1/705C12Q2521/101C12Q2527/101C12Q2565/625
Inventor 勾红潮蔡汝健李春玲蒋智勇宋帅李淼李艳楚品品杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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