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Multiplex PCR detection kit for identifying porcine circovirus

The technology of a porcine circovirus and a detection kit is applied in the field of molecular biology of animal viruses, which can solve the problems that porcine circoviruses are difficult to meet actual needs and have little research, and achieve reduced detection costs and detection time, strong specificity, and reliability good repeatability

Inactive Publication Date: 2018-08-28
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are relatively few studies on PCV3, and the original detection and typing methods for porcine circovirus (PCV1 and PCV2) have been difficult to meet the actual needs

Method used

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  • Multiplex PCR detection kit for identifying porcine circovirus
  • Multiplex PCR detection kit for identifying porcine circovirus
  • Multiplex PCR detection kit for identifying porcine circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the establishment of PCV multiplex PCR method

[0037] 1. Primer design

[0038] According to the PCV1, PCV2 and PCV3 sequences included in GenBank, by comparing the differences between PCV1, PCV2 and PCV3, and combining the inventor's experience in studying porcine circovirus for many years, we designed three pairs of specific primers, as follows:

[0039] PCV1-F: 5'-GAAAGTGAGCGGGAAGAT-3', as shown in SEQ ID NO: 1;

[0040] PCV1-R: 5'-CTGATTGCTGGTAATCAA-3', as shown in SEQ ID NO: 2;

[0041] PCV2-F: 5'-CACATCGAGAAAGCGAAAGGAAC-3', as shown in SEQ ID NO: 3;

[0042] PCV2-R: 5'-TGCGGGCCAAAAAAGGTACAGTT-3', as shown in SEQ ID NO: 4;

[0043] PCV3-F: 5'-AGCAGTGCTCCCCATTGA-3', as shown in SEQ ID NO: 5;

[0044] PCV3-R: 5'-TGGGCCCGACCAAATCCGG-3', as shown in SEQ ID NO:6.

[0045] Using the above three pairs of primers for PCR amplification, the predicted amplified fragment size of PCV1 is 310bp, the predicted amplified fragment size of PCV2 is 505bp, and the...

Embodiment 2

[0064] Embodiment 2: the specificity test of PCV multiplex PCR method

[0065] Detect PCV1 with the PCV multiplex PCR method that embodiment 1 establishes, PCV2, PCV3 and 8 other porcine source virus strains, comprise swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine encephalitis virus (JEV), rotavirus (RV), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV) to validate the method Test the specificity of different samples.

[0066] The results showed that only PCV1, PCV2 and PCV3 were amplified to obtain fragments of 310bp, 505bp and 1021bp, respectively, and no amplified bands were produced by other viruses. Experimental result confirms, primer of the present invention and detection method have very high specificity ( figure 2 ).

Embodiment 3

[0067] Embodiment 3: the sensitivity test of PCV multiplex PCR method

[0068] The PCV multiplex PCR method established in Example 1 was used to detect different amounts of DNA of PCV1, PCV2 and PCV3 to detect the sensitivity of the method.

[0069] The results of PCV multiplex PCR amplification detection of different dilution samples image 3 . The results showed that the sensitivity of the PCV composite PCR was 10ng / μL.

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Abstract

The invention discloses a multiplex PCR detection kit for identifying porcine circovirus (PCV). The kit comprises the following three pairs of multiplex PCR detection primers which can quickly distinguish genotype PCV1, genotype PCV2 and genotype PCV3: PCV1-F and PCV1-R, PCV2-F and PCV2-R, and PCV3-F and PCV3-R.The multiplex PCR detection kit disclosed by the invention can be used for detecting the PCV and distinguishing different genotypes, has the characteristics of being quick, simple, convenient, high in specificity and high in sensibility, and can perform large batches of sample detectionat the same time; only one strand displacement amplification is needed, so that the situation whether a sample is infected by PCV1, PCV2 or PCV3, or is jointly infected by two or three of the PCV1, the PCV2 and the PCV3 can be identified; and the multiplex PCR detection kit can provide technical support for epidemic situation surveillance, epidemiologic investigation and comprehensive preventingand controlling of the PCV, and has a favorable application prospect.

Description

technical field [0001] The invention belongs to the technical field of animal virus molecular biology, in particular to a multiplex PCR detection kit for identifying and detecting porcine circovirus (porcine circovirus, PCV). Background technique [0002] Porcine circovirus (PCV) belongs to the genus Circovirus (Circoviridae) in taxonomy, and is one of the smallest known animal viruses. Virus particles have a diameter of 14-17nm, a 20-sided symmetrical structure, no envelope, and contain a covalently closed single-strand circular negative-strand DNA. The genome size is about 1.76kb. Before 2016, only two genotypes of porcine circovirus (PCV), PCV1 and PCV2, were found worldwide. PCV1 is a PK-15 pollutant in pig kidney cells, but it does not produce cytopathic effects and is not pathogenic to pigs. PCV2 is the main pathogen that causes multisystemic wasting syndrome in weaned piglets, and is closely related to porcine dermatitis and nephrotic syndrome, porcine respiratory d...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 杨克礼田永祥段正赢郭锐周丹娜袁芳艳刘泽文刘威高婷
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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