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LAMP detection primer for distinguishing African swine fever virus wild strain from MGF360-505R gene deleted strain and kit thereof

A technology for African swine fever virus and gene deletion, applied in the field of LAMP detection primers and kits, can solve the problems of inability to distinguish virus infection or vaccination, inconvenient conventional nucleic acid detection methods, and inability to realize on-site detection, etc., achieving scientific primer design , high sensitivity and high specificity

Active Publication Date: 2020-03-17
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the first aspect of the present invention is aimed at the technical problem that the common detection method of African swine fever virus cannot distinguish between virus infection or vaccination, but the conventional nucleic acid detection means are inconvenient, the dependence on the instrument is relatively strong, and the on-site detection cannot be realized. , providing a LAMP detection primer set for distinguishing African swine fever virus wild strains from MGF360-505R gene deletion strains

Method used

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  • LAMP detection primer for distinguishing African swine fever virus wild strain from MGF360-505R gene deleted strain and kit thereof
  • LAMP detection primer for distinguishing African swine fever virus wild strain from MGF360-505R gene deleted strain and kit thereof
  • LAMP detection primer for distinguishing African swine fever virus wild strain from MGF360-505R gene deleted strain and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Primer Design

[0064] 1. Plasmid

[0065] According to the African swine fever virus gene sequence (GenBank: MK333180.1) provided on Genebank, a part of the fragment (shown in SEQ ID No.11) was selected, and the plasmid pUC57-VIR was synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd. Dissolve and dilute to 1.0 x 10 6 copies / μL.

[0066] 2. Identification of positive plasmids

[0067] Construct a positive plasmid, construct a plasmid DNA containing a fragment of the target gene P72 (B646L) and a plasmid DNA missing the fragments on both sides of the 27942-35500 position, the sequence of the target gene P72 (B646L) fragment is shown in SEQ ID No.13 , the sequence of the region fragment on both sides of the deletion at position 27942-35500 is shown in SEQ ID No.14.

[0068] Target gene P72(B646L) fragment:

[0069] TACTGTAACGCAGCACAGCTGAACCGTTCTGAAGAAGAAGAAAGTTAATAGCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGG...

Embodiment 2

[0099] The establishment of embodiment 2LAMP reaction system

[0100] Determine the content and ratio of each component in the 25 μl reaction system detected by LAMP, and place it in a constant temperature container for amplification. The test result can be judged by combining the white turbidity with naked eyes and gel electrophoresis. The 25 μL reaction system is shown in Table 2.

[0101] Table 2 25 μ LAMP reaction system

[0102] name Dosage Bst DNA polymerase 1.0 μL 10×Bst buffer 2.5μL dNTP 2.5μL Mg2+ / Mn2+ mixture 2.0 μL Primer 6.0 μL DNA template 2.0 μL ddH2O 9.0μl Total 25.0 μL

[0103] Set the concentration to 10 3 The copied positive plasmid was used as the detection object, and the LAMP reaction temperature was determined to be 65° C., and the optimal reaction time was 50 minutes.

[0104] The results showed that the reaction was carried out at 65°C for 50 minutes. It can be seen from the res...

Embodiment 3

[0105] Embodiment 3 specificity test

[0106] Use conventional methods to extract African swine fever virus nucleic acid, swine fever virus nucleic acid, porcine reproductive and respiratory syndrome virus nucleic acid, porcine circovirus type 2 nucleic acid, porcine erysipelas virus nucleic acid extract as a template, using LAMP detection primers in Example 1, The detection was carried out according to the reaction system and reaction conditions in Example 2 to detect the specificity of the primers. The results are shown in Table 3, attached Figure 4

[0107] Table 3 Specificity determination statistics of LAMP

[0108]

[0109]

[0110] The experimental results showed that the kit was used to detect the extracts of classical swine fever virus nucleic acid, porcine reproductive and respiratory syndrome virus nucleic acid, porcine circovirus type 2 nucleic acid, and porcine erysipelas virus nucleic acid, all of which were negative. The first set of primers detected t...

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Abstract

The invention discloses an LAMP detection primer for distinguishing an African swine fever virus wild strain from an MGF360-505R gene deleted strain and a kit thereof. The LAMP detection primer comprises two sets of primers that are used for a conservative capsid protein gene P72 (B646L) sequence of an African swine fever virus wild strain and two sides of the 27942-35500th deletion region of theAfrican swine fever MGF360-505R gene deleted strain respectively so as to specifically detect the African swine fever MGF360-505R gene deleted strain. With the constant-temperature reaction detectionmeans, the whole reaction can be realized only by heating and the detection result can be directly visually inspected, so that the LAMP detection primer and the kit are suitable for field detection; the method is high in detection sensitivity, strong in specificity, high in repeatability and high in detection speed and can be used as a basic detection means for effectively identifying the Africanswine fever virus wild strain and the MGF360-505R gene deletion strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a LAMP detection primer and a kit for distinguishing African swine fever virus wild strains and gene deletion strains. Background technique [0002] African swine fever is an acute, hemorrhagic and severe infectious disease caused by African swine fever virus infection of domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.). The clinical manifestations of affected pigs are fever (up to 40-42°C), rapid heartbeat, dyspnea, partial cough, serous or mucopurulent secretions in the eyes and nose, cyanosis of the skin, obvious bleeding of lymph nodes, kidneys, and gastrointestinal mucosa. The most acute and acute infections have a mortality rate of up to 100%. Domestic pigs, wild boars and soft ticks are the natural hosts of African swine fever. African swine fever can be transmitted directly between domestic pigs and wild boars, or t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/125Y02A50/30
Inventor 沈永义王瑞晨陈瑞爱杨柔
Owner SOUTH CHINA AGRI UNIV