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Dual PCR primer, detection method and kit for detecting porcine circovirus type 2 and circovirus type 3

A porcine circovirus and detection kit technology, applied in the biological field, can solve the problems of prone to false positives, low sensitivity, uneven detection sensitivity and specificity, etc.

Inactive Publication Date: 2018-04-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, virus isolation and serological detection methods are time-consuming, low-sensitivity and prone to false positives
The PCR detection method is mainly based on a single detection of a certain virus type, and combined PCR detection of multiple virus types cannot be performed, and the sensitivity and specificity of detection are also uneven.

Method used

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  • Dual PCR primer, detection method and kit for detecting porcine circovirus type 2 and circovirus type 3
  • Dual PCR primer, detection method and kit for detecting porcine circovirus type 2 and circovirus type 3
  • Dual PCR primer, detection method and kit for detecting porcine circovirus type 2 and circovirus type 3

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Screening of specific primers and system optimization of porcine circovirus type 2 and type 3 duplex PCR method

[0032] 1) Design of dual PCR primers for porcine circovirus type 2 and type 3

[0033] According to the gene sequences of two pathogenic bacteria, porcine circovirus type 2 (Porcine circovirus 2) and porcine circovirus type 3 (Porcine circovirus 3) published by GeneBank, their highly conserved specific sequences were obtained through sequence analysis, and according to the specificity Specific primers were designed for conserved sequences using Primer5.0 software. The primers were all synthesized in Beijing Ruibo Xingke Biotechnology Co., Ltd., and the specific sequences are shown in Table 1:

[0034] Table 1 is directed at the specific primers of porcine circovirus type 2 and porcine circovirus type 3:

[0035]

[0036] 2) Reaction system and reaction conditions for double PCR of porcine circovirus type 2 and type 3

[0037] The reaction system of porci...

Embodiment 2

[0049] Performance verification of the kit of the present invention

[0050] 1) Specificity test

[0051] Porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine senega valley virus (SVV), porcine blue ear disease virus (PRRSV), porcine D-coronavirus The virus (PDCoV) was amplified, and sterilized water was selected as a negative control to test the specificity of the established method.

[0052] The result is as Figure 4 As shown, the target fragment size of porcine circovirus type 2 is 511bp; the target fragment size of porcine circovirus type 3 is 701bp; Strips, respectively 511bp and 701bp; TGEV, PEDV, SVV, PRRSV, PDCoV virus no specific band amplification.

[0053] 2) Sensitivity test

[0054] The 511bp fragment amplified by porcine circovirus type 2 and porcine circovirus type 3 gene were connected to the pMD19-T vector, transformed into Escherichia coli DH5a, and positive colonies were picked and cultured, using a plasmid...

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Abstract

The invention provides a dual PCR primer, detection method and kit for detecting porcine circovirus type 2 and circovirus type 3. The dual PCR primer is designed according to a highly-conserved specific sequence for detecting porcine circovirus type 2 and circovirus type 3, and the detection method for monotube synchronous detecting of porcine circovirus type 2 and circovirus type 3 is established. The kit has the advantages of quickness, simplicity, convenience and high specificity, sensitivity and reliability, sample analysis on a large scale can be performed at the same time, whether a sample is infected with porcine circovirus type 2 or the porcine circovirus type 3 or the two can be identified and diagnosed through single reaction, powerful technical support is provided for monitoring, preventing and controlling porcine circovirus epidemic, and the kit has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double PCR primer, a detection method and a kit for detecting porcine circovirus type 2 and type 3. Background technique [0002] Porcine circovirus (porcine circovirus, PCV) is a small, non-enveloped, icosahedral symmetrical, circular, covalently closed single-stranded DNA virus belonging to the family Circoviridae. Virus particles have an average diameter of 17nm and replicate in a rolling circle manner, which is the smallest non-enveloped DNA virus found so far. Brain, Allan and others believed that porcine circovirus can be divided into two genotypes according to the pathogenicity, antigenicity and nucleotide sequence of porcine circovirus, namely porcine circovirus type 1 (PCV1) and porcine circovirus Type 2 (PCV2). PCV1 was first identified as a contaminant in PK cell cultures in 1974 and is not pathogenic in pigs. PCV2 was first reported in 1998. It can cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 贺东生俞丽苏丹萍徐帅飞梁伟放
Owner SOUTH CHINA AGRI UNIV
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