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Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and applications

A technology of porcine circovirus and type 3 virus, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., which can solve the problem of limited application range, complex processing, and inability to identify and detect two viruses at the same time And other issues

Active Publication Date: 2021-08-31
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have disadvantages such as high cost, need for specific equipment, time-consuming and complex sample processing, and are generally aimed at a single pathogen, unable to identify and detect two viruses at the same time, which seriously limits their practical application range

Method used

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  • Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and applications
  • Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and applications
  • Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Establishment of a dual RAA detection method for porcine circovirus type 2 and porcine circovirus type 3.

[0083] 1) Design and screening of embodiment 1, PCV2 and PCV3 primers and probes

[0084] At present, there is no special procedure for the design of RAA primers and probes, and the primers and probes in the test need to be optimized and screened. In this study, 2 pairs of primers and 2 probes were screened out, and a real-time fluorescent RAA detection method for PCV2 and PCV3 was established. details as follows:

[0085] (1) Design of primers and probes

[0086] The inventor analyzed and determined through literature search that the specific sequences of PCV2 and PCV3 used in the present invention are the target genes. Obtain the known template gene sequence from the NCBI database, and screen out a sequence of the PCV2 conserved region Cap genome (MH931449.1), which contains a 758-base nucleotide fragment with the nucleotide shown in SEQ ID NO.7 Ac...

Embodiment 2

[0104] Embodiment 2: Optimization of RAA reaction system, amplification and detection conditions

[0105] (1) Primer probe concentration

[0106] Set the concentration of each primer to 10 μmol / L, and the concentration gradient of the probe to 10 μmol / L, 5 μmol / L, and 2.5 μmol / L, and combine one concentration of primers with three concentrations of probes to form 3 groups of combinations (see Table 4), and each group of combinations is provided with a group of negative controls. RAA amplification was carried out at 39°C, and after the amplification was completed, the combination with the best amplification effect was screened.

[0107] Table 4 Combination numbers of reverse primer concentration and probe concentration

[0108] Numbering combination Amplification efficiency 1 10μM(P3-F2+P3-R1+P2-F3+P2-R2)+10μM(P2-P1+P3-P1) + 2 10μM(P3-F2+P3-R1+P2-F3+P2-R2)+5μM(P2-P1+P3-P1) +++ 3 10μM(P3-F2+P3-R1+P2-F3+P2-R2)+2.5μM(P2-P1+P3-P1) ++

[010...

Embodiment 3

[0141] Example 3 Specificity test of the dual real-time fluorescence RAA kit.

[0142] The extracted viral RNAs such as FMDV, SVDV, PRRSV, CSFV, etc. were reverse-transcribed to synthesize cDNA, and stored for future use. The cDNA of FMDV, SVDV, PRRSV, CSFV and the DNA samples of PCV2 and PCV3 are detected by the double real-time fluorescent RAA kit of the present invention to determine the specificity of the double real-time fluorescent RAA detection method of the present invention.

[0143] The result is as image 3 shown. The test results show that the present invention can simultaneously detect porcine circovirus type 2 and porcine circovirus type 3 viruses, and other viral nucleic acids have no amplification curves, indicating that the dual real-time fluorescent RAA kit of the present invention has good specificity.

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Abstract

The invention discloses real-time fluorescent RAA primers and probes for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses, a kit including the primers and probes, and applications. The sensitivity of the method is 102 copies / reaction; and each reaction may simultaneously and minimally detect 102 copies of porcine circovirus type 2 viruses and 102 copies of porcine circovirus type 3 viruses. Therefore, the porcine circovirus type 2 and porcine circovirus type 3 viruses can be rapidly, efficiently, sensitively and simultaneously detected; and effective technological means can be provided for rapid identification detection of mixed infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of biomedical preventive veterinary inspection, and specifically relates to a kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and an application thereof. Background technique [0002] Porcine circovirus type 2 and porcine circovirus type 3 are two important pathogens affecting the pig industry in my country and the world. The genome structure of PCV2 and PCV3 is similar, mainly including two main proteins, Rep and Cap, but the homology of the two Cap proteins is only 36%-37%. Porcine circovirus type 2 has strong pathogenicity, and it mostly infects piglets aged 5 to 12 weeks, causing weaned piglet multisystemic wasting syndrome (PMWS), porcine dermatitis and nephrotic syndrome, PCV2-related pneumonia, reproductive disorders and Enteritis, etc., the mortality rate is higher. Porcine circovirus type 3 PCV3 is a newly discover...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2561/113C12Q2563/107
Inventor 吴江林彦星花群义黄超华史卫军曹琛福曾少灵杨俊兴阮周曦刘建利
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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