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ASFV detection kit and primer probe composition and application thereof

A kit and composition technology, applied in the biological field, can solve the problems of no drugs that can effectively inhibit the African swine fever virus, no vaccines, etc., and achieve the effect of eliminating false positive events, important economic benefits and social value

Active Publication Date: 2021-12-28
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex immune escape mechanism and large genetic structure of ASFV, there is currently no safe and efficient commercial vaccine, nor is there any drug that can effectively inhibit African swine fever virus

Method used

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  • ASFV detection kit and primer probe composition and application thereof
  • ASFV detection kit and primer probe composition and application thereof
  • ASFV detection kit and primer probe composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 detects the source and preparation of samples

[0057] The plasmid pUC57 in this example was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0058] The highly conserved region of African swine fever virus (ASFV) P17 gene sequence after screening (African swine fever virus-specific p17 gene fragment) is representative of ASFV-specific sequences and can be used for nucleic acid identification of various African swine fever virus strains Specific identification of positive templates.

[0059]The present invention uses the recombinant plasmid (plasmid pUC57-p17) carrying the African swine fever virus-specific p17 gene fragment as a detection sample. The recombinant plasmid pUC57-p17 is to replace the fragment (small fragment) between the EcoRV and BamHI recognition sites of pUC57 with the p17 gene fragment whose nucleotide sequence is SEQ ID No.31, and keep other nucleotide sequences of pUC57 unchanged The obtained recombinant vector.

Embodiment 2

[0060] The design of embodiment 2 primer, probe, crRNA

[0061] 1. Design of primers and probes

[0062] Aiming at the p17 gene fragment of the highly conserved region of African swine fever virus, design a specific primer pair for detecting African swine fever virus and a probe FAM-N-BHQ2 that specifically recognizes the p17 gene fragment of the highly conserved region of African swine fever virus sequence , primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0063] The primer pairs are as follows:

[0064] A1) consists of primer ASF-RPA-F1 and primer ASF-RPA-R1; the primer ASF-RPA-F1 is a single-stranded DNA molecule shown in SEQ ID No.1; the primer ASF-RPA-R1 is SEQ ID The single-stranded DNA molecule shown in No.2;

[0065] A2) consists of primer ASF-RPA-F1 and primer ASF-RPA-R2; the primer ASF-RPA-F1 is a single-stranded DNA molecule shown in SEQ ID No.1; the primer ASF-RPA-R1 is SEQ ID The single-stranded DNA molecule shown in No.3.

...

Embodiment 3

[0076] Example 3 Detection of ASFV based on RPA technology and Cas12a enzyme digestion technology

[0077] RPA amplification system, CRISPR / Cas12a detection system, RPA amplification program are shown in Table 1, Table 2 and Table 3:

[0078] Table 1 Solution A reaction system (96-well PCR plate + 10% reagent loss)

[0079]

[0080] Note: Buffer A and Buffer B are from the DNA Constant Temperature Rapid Amplification Kit, the product of Nanjing Wobo Biotechnology Co., Ltd., kit number: WLB8201KIT.

[0081] Table 2 Solution B reaction system (96-well PCR plate + 10% reagent loss)

[0082] Reagent concentration Volume (μl) Cas12a 250-1000nM 1 2.1 NEB buffer 10x 5.0 h 2 o

13 FAM-N-BHQ 2

200-1000nM 2 p17-crRNA-2 500-1000nM 4 / hole DNA template / 1 / hole total 25 / hole

[0083] Cas12a in Table 2 was purchased from NEB Company.

[0084] Table 3 RPA amplification program

[0085] temperat...

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Abstract

The invention discloses an ASFV detection kit as well as a primer probe composition and application thereof. The invention specifically discloses a kit for detecting African swine fever virus. The kit comprises a primer ASF-RPA-F1 and a primer ASF-RPA-R1 or a primer ASF-RPA-R2, and also comprises at least any one type of p17-crRNA as shown in SEQ ID No.4-29, and a probe FAM-N-BHQ2. An established rapid, accurate, visual and low-cost African swine fever virus detection method combines an RPA technology and a Cas12a enzyme digestion technology, can efficiently, sensitively, specifically and accurately judge whether a sample to be detected contains the African swine fever virus or not, provides a powerful detection tool for strictly controlling the propagation of ASFV, and has important economic benefits and social values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an ASFV detection kit and its primer probe composition and application, in particular to a special detection kit for accurate, simple, sensitive and visual detection of African swine fever virus (ASFV) nucleic acid and its primers Probe composition and detection method. Background technique [0002] African swine fever virus (ASFV) is a large nucleoplasmic DNA virus that can infect domestic pigs and various wild boars and cause acute, hemorrhagic, and severe infectious diseases with a mortality rate of up to 100%. Due to the complex immune escape mechanism and large gene structure of ASFV, there is currently no safe and efficient commercial vaccine, nor is there any drug that can effectively inhibit ASF virus. Therefore, the prevention and detection of African swine fever is particularly important. [0003] Recombinase Polymerase Amplification (RPA, Recombinase Polymerase Amplificati...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2563/107C12Q2525/161C12Q2521/327Y02A50/30
Inventor 唐中林刘思远唐义杰陈慕雅
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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