Multiplex PCR diagnosis method capable of quickly distinguishing porcine circovirus 1 type (PCV1) and 2 type (PCV2) and special-purpose kit

A technology for porcine circovirus and circovirus, which is applied in the fields of diagnosis, multiplex PCR diagnostic methods and special kits, can solve problems such as contamination of PCV1 and economic losses in the pig industry, and achieve strong specificity.

Inactive Publication Date: 2015-01-07
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

[0003] The purpose of the present invention is to aim at the current situation that PCV2 infection is ubiquitous in my country, which brings serious economic losses to the pig industry, and PCV1 is often polluted in the culture process of PCV2, and provides a method that can facilitate the laboratory to carry out cell culture and PCV2 cytotoxic purification. Diagnostic methods and dedicated kits for on-the-go monitoring

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  • Multiplex PCR diagnosis method capable of quickly distinguishing porcine circovirus 1 type (PCV1) and 2 type (PCV2) and special-purpose kit
  • Multiplex PCR diagnosis method capable of quickly distinguishing porcine circovirus 1 type (PCV1) and 2 type (PCV2) and special-purpose kit
  • Multiplex PCR diagnosis method capable of quickly distinguishing porcine circovirus 1 type (PCV1) and 2 type (PCV2) and special-purpose kit

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Embodiment Construction

[0010] 1.1 Primer Design: According to the difference between the PCV1 and PCV2 gene sequences published on NCBI, three specific primers L1, L2, and L3 were designed to amplify porcine circovirus type II (PCV2). The estimated amplified fragment size of porcine circovirus type 1 (PCV1) is 198bp.

[0011] L1: 5′-TATTACCGGCGCACTTCGGCAGCG-3′

[0012] L2: 5′-TCACTCCGTTGTCCCTGAGATCT-3′

[0013] L3: 5′-TCTTCCAAACCTTCCTCTCCGCAA-3′

[0014] Among them, L1 and L2 can specifically amplify porcine circovirus type II (PCV2), and the estimated amplified fragment size is 404bp, and L1 and L3 amplify porcine circovirus type I (PCV1), and the estimated amplified fragment size is 198bp, annealed The temperature was 55°C and 30 cycles were used.

[0015] 1.2 Extraction of viral DNA

[0016] The culture lysates of PCV1 and PCV2 cells were taken respectively, and after repeated freezing and thawing three times, they were centrifuged at 5000 g for 15 minutes.

[0017] 1) Take 500 μL of superna...

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Abstract

The invention patent relates to a diagnosis technology in the technical field of agriculture. Specific primers L1, L2 and L3 are designed automatically, and virus DNAs are extracted; on the basis, the PCR method is used for discriminating and detecting different serotypes of the porcine circovirus, the size of an estimated amplified fragment of the amplified porcine circovirus II type (PCV2) is 404bp, and the size of an estimated amplified fragment of the amplified porcine circovirus I type (PCV1) is 198bp; on the basis, a special-purpose kit is successfully developed. Compared with the prior art, the diagnosis method and the special-purpose kit have stronger specificity and sensibility, and compared with methods of virus separation, IFA and the like, the coincidence rate of the method can reach more than 90%. The amplified results of the primers for a porcine reproductive and respiratory syndrome virus, a hog cholera virus, a porcine pseudorabies virus, porcine parvovirus are all feminine, and the primers can be widely used for detecting the porcine circovirus in clinical applications and laboratories.

Description

technical field [0001] The patent of the present invention relates to diagnostic technology in the field of veterinary biotechnology, specifically a multiplex PCR diagnostic method and a special kit capable of quickly distinguishing porcine circovirus type 1 (PCV1) and type 2 (PCV2). Background technique [0002] Porcine circovirus (PCV) is a small, icosahedral symmetric, non-enveloped, single-stranded circular DNA virus. The diameter of the virus particle is about 17nm, which is one of the animal viruses with the smallest DNA found so far. Circoviruses have two genotypes: PCV1, which is nonpathogenic, and PCV2, which is pathogenic. Porcine circovirus type 2 is also the main pathogen that causes multisystemic weakness syndrome (PMWS) in weaned piglets. It brings huge economic losses to the breeding industry and is one of the important diseases that endanger the world's pig industry at present. Although porcine circovirus type 1 is not pathogenic, it often contaminates pig...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/112C12Q2600/16
Inventor 李俊王金宝时建立徐绍建彭喆丛晓燕黄保华吴晓燕刘畅
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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