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Amplification primer pair for detecting African swine fever virus, detection kit and application

An African swine fever virus and amplification primer technology, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of long time required for detection, and achieve simple and fast interpretation of results, high sensitivity and sensitivity high effect

Pending Publication Date: 2022-06-28
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods either require a long time for detection, or have certain requirements for the target sequence and length, and have certain restrictions on primer design

Method used

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  • Amplification primer pair for detecting African swine fever virus, detection kit and application
  • Amplification primer pair for detecting African swine fever virus, detection kit and application
  • Amplification primer pair for detecting African swine fever virus, detection kit and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Design of Rapid Amplification Primers Based on African Swine Fever Virus B646L Gene

[0046] According to the complete genome sequence of African swine fever virus and other swine viruses with similar clinical symptoms (MW788405_ASFV, AF176348_PRRSV, KT119352_CSFV, M34651_PRV, NC001718_PPV, AY181948_PCV2, NC003436_PEDV, MN198861_SIV) published in GenBank, the gene sequence database of the American Center for Biotechnology Information, use Molecular biology tools such as BLASTn, MAFFT, DNASTAR and other molecular biology tools were used to conduct multiple sequence alignment analysis of the whole genome, and obtained the conserved region specific to the African swine fever virus B646L gene. Based on conserved target gene sequences, Primer Premier5.0 and online tool NUPACK (http: / / www.nupack.org / ) were used to design primers for rapid amplification. The designed primers were further verified by BLASTn and Primer-BLAST searches. The primer information is as follows:

[00...

Embodiment 2

[0052] Establishment of a rapid fluorescence detection method for African swine fever virus

[0053] The African swine fever virus (ASFV) nucleic acid standard substance (5.8×10 3 copies / μL) (purchased from Guangzhou Bang Desheng Biotechnology Co., Ltd.) and negative control (sterilized saline) for amplification method testing to obtain the optimal reaction system and reaction temperature.

[0054] 1. Optimization of the reaction system

[0055] In order to verify the effect of graphene, the reaction system in this experiment is divided into two groups:

[0056] Graphene-free group: with African swine fever virus (ASFV) nucleic acid standard material (original concentration of 5.8 × 10 3 copy / μL) as template, 20μL reaction system includes: 2μL 10× amplification buffer, 2μL dNTPs (1~10mM), 1μL liquid polyethylene glycol, 1μL betaine (1~10mM), primers F and R ( 10 μM) 2 μL each, 0.8 μL Bst DNA polymerase (8000 U / mL), 1 μL Eva Green (20×), 5 μL template and 2.2 μL enzyme-free ...

Embodiment 3

[0067] Repeatability test of rapid fluorescent detection method for African swine fever virus

[0068] Using African swine fever virus (ASFV) nucleic acid standard material (the original concentration is 5.8 × 10 3 copy / μL) as template, 20μL reaction system includes: 2μL 10× amplification buffer, 2μL dNTPs (1~10mM), 1μL liquid polyethylene glycol, 1μL betaine (1~10mM), primers F and R ( 10 μM) 2 μL each, 0.8 μL Bst DNA polymerase (8000 U / mL), 1 μL graphene dispersion (1–10 ng / μL), 1 μL Eva Green (20×), 5 μL template and 1.2 μL enzyme-free water. The reaction system was prepared in a micro reaction tube, and a negative control was set at the same time. Mix the reaction tube containing the reaction system, centrifuge briefly, and place the reaction tube in a fluorescence quantitative PCR instrument. The reaction program was: 73-74°C for 1 second, 60°C for 1 second, a total of 45 cycles.

[0069] The result is as Image 6 As shown in Table 2, by performing 10 amplification te...

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Abstract

The invention discloses an amplification primer pair for detecting African swine fever virus, a detection kit and application, the amplification primer pair is designed based on a B646L gene specificity conserved region of the African swine fever virus, and comprises a forward primer and a reverse primer; the sequence of the forward primer is as shown in SEQ ID NO: 1, and the sequence of the reverse primer is as shown in SEQ ID NO: 2; the kit disclosed by the invention consists of a virus nucleic acid rapid amplification reagent. The virus nucleic acid rapid amplification reagent comprises a reaction buffer solution containing graphene, an African swine fever virus B646L gene rapid amplification primer and a quality control product. By means of a fluorescent quantitative PCR instrument, fluorescent quantitative detection of the African swine fever virus can be completed within 30 minutes by means of the method. The African swine fever virus detection method is simple and convenient to operate, safe, high in sensitivity, strong in specificity, good in repeatability and beneficial to practical application.

Description

technical field [0001] The invention relates to an amplification primer for detecting African swine fever virus, in particular to an amplification primer pair, detection kit and application based on African swine fever virus B646L gene. Background technique [0002] African swine fever is an acute, severe and highly contagious infectious disease caused by African swine fever virus (ASFV), which has brought a huge threat to the global pig industry. This infectious disease belongs to the notifiable animal disease of the World Organization for Animal Health (OIE), and it is also a type of animal disease stipulated by China. African swine fever is characterized by a short course of onset, the most acute and acute infections have a mortality rate of up to 100%, and clinical manifestations include fever (40-42°C), rapid heartbeat, dyspnea, partial cough, and serous or mucous eyes and nose. Purulent secretions, cyanosis of the skin, and obvious hemorrhage of lymph nodes, kidneys, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2527/125C12Q2563/107
Inventor 杨剑波庄林林朱孟玲施一成吴井生宋春雷孙丽谢海强赵彬任希艳
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY