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Kit for rapidly and visually detecting PCV3 and detection method thereof

A PCV3-RPA-F, PCV3-RPA-R technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effect of simple kit, accurate test results and cost saving

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

AI Technical Summary

Problems solved by technology

Same as PCV2, the Cap protein of PVC3 can induce autophagy in host cells, and can also suppress the host's natural immune response. Since there is no safe and efficient PCV3 vaccine at present, seroepidemiological investigations can more quickly from larger Therefore, the early prevention, control and detection of PCV3 is particularly important

Method used

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  • Kit for rapidly and visually detecting PCV3 and detection method thereof
  • Kit for rapidly and visually detecting PCV3 and detection method thereof
  • Kit for rapidly and visually detecting PCV3 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Acquisition of a plasmid sample carrying PCV3 conserved virus sequence information

[0054] The highly conserved region of porcine circovirus type 3 virus (PCV3) cap gene sequence after screening (PCV3 specific cap gene fragment, SEQ ID No.17) has the representativeness of PCV3 specific sequence, and the present invention is used to carry PCV3 specific The recombinant plasmid (plasmid pUC57-cap413) of cap gene fragment was used as the detection sample (plasmid sample carrying PCV3 conservative virus sequence information). The recombinant plasmid pUC57-cap413 is to replace the fragment (small fragment) between the EcoRI and HindIII restriction endonuclease recognition sites of the pUC57 vector with the DNA fragment shown in SEQ ID No.17 in the sequence listing, keeping the pUC57 vector The other nucleotide sequences remain unchanged, resulting in a recombinant vector.

[0055] The nucleic acid information fragment carrying the PCV3 conservative virus sequence ...

Embodiment 2

[0056] The design of embodiment 2 primers, probes and crRNA

[0057] 1. Design of primers and probes

[0058] Aiming at the nucleic acid fragment of the highly conserved region of the cap gene of porcine circovirus type 3 virus (PCV3), the specific primer pair for detecting PCV3 and the probe FAM-N-BHQ2 for specifically recognizing the cap gene fragment of PCV3, primers and probes are designed. Needles were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0059] The primer pair is composed of forward primer PCV3-RPA-F and reverse primer PCV3-RPA-R; the forward primer PCV3-RPA-F is a single-stranded DNA molecule shown in SEQ ID No.1; the The reverse primer PCV3-RPA-R is a single-stranded DNA molecule shown in SEQ ID No.2; the primer pair can be used to specifically amplify the cap gene fragment specific to PCV3; the nucleotide sequence of the cap gene fragment As shown in SEQ ID No.17.

[0060] The nucleotide sequence of the probe FAM-N-BHQ2 is shown in SEQ ID No....

Embodiment 3

[0071] Example 3 Detection of PCV3 based on RPA technology and Cas12a enzyme digestion technology

[0072] The reaction system and amplification program in this embodiment are shown in Table 2-7:

[0073] Table 2 Solution A reaction system (96-well PCR plate + 10% reagent loss)

[0074] Reagent concentration Volume (μl) Buffer A 14.55 Buffer B 1.25 h 2 o

NA 6.2 Primer PCV3-RPA-F 10-20μM 1 Primer PCV3-RPA-R 10-20μM 1 DNA template ng / μl 1 / hole total 25 / hole

[0075] Table 3 Solution A Alone (RPA) Amplification Program

[0076] temperature(°C) time (seconds) Number of cycles (pieces) 37 1200-1500 1

[0077] Note: Buffer A and Buffer B come from the DNA Constant Temperature Rapid Amplification Kit, a product of Nanjing Wobo Biotechnology Co., Ltd., kit number: WLB8201KIT.

[0078] Table 4 Solution B reaction system (96-well PCR plate+10% reagent loss)

[0079] Reag...

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Abstract

The invention discloses a kit for rapidly and visually detecting PCV3 and a detection method thereof. According to the kit for detecting PCV3 and the detection method thereof, the kit comprises a primer PCV3-RPA-F and a primer PCV3-RPA-R, and also comprises at least any one kind of capp-crRNA as shown in SEQ ID No.3-15, and a probe FAM-N-BHQ2. The kit is good in specificity, and the detection sensitivity of the kit can reach a single copy level. An established rapid, accurate, visual and low-cost PCV3 detection method combines an RPA technology and a Cas12a enzyme digestion technology, can simply, efficiently, sensitively, specifically and accurately judge whether a sample to be detected contains PCV3 or not, provides a powerful detection tool for strictly controlling the propagation of PCV3, and has important economic benefits and social values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kit for rapid and visual detection of PCV3 and a detection method thereof, in particular to an accurate, simple, sensitive and visual detection of porcine circovirus type 3 virus combined with RPA technology and Cas12a enzyme digestion technology (PCV3) nucleic acid special detection kit, primer probe, crRNA, and detection method thereof. Background technique [0002] Porcine circovirus type 3 (PCV3) is a non-enveloped single-stranded circular DNA virus with a full length of 2000bp and less than 40% homology with PCV1 / 2. Genetic evolution analysis is located in different branches . The PCV3 genome includes 3 open reading frames, of which ORF2 is mainly responsible for encoding capsid protein (capsid protein, Cap), which is the only structural protein, contains antigen reactive clusters, and can induce the body to produce neutralizing antibodies. PCV3 and porcine dermatitis nephrotic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/507C12Q2521/301C12Q2537/1376C12Q2522/101C12Q2563/107
Inventor 唐中林刘思远唐义杰陈慕雅
Owner AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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