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Real-time fluorescent quantitative PCR primer, kit and detection method for detecting porcine circovirus 3

A real-time fluorescence quantitative and porcine circovirus technology, applied in the field of molecular biology, achieves high sensitivity, stable repeatability, and strong specificity

Inactive Publication Date: 2017-11-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there is no report on the application of fluorescent quantitative PCR method to detect PCV3 in China

Method used

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  • Real-time fluorescent quantitative PCR primer, kit and detection method for detecting porcine circovirus 3
  • Real-time fluorescent quantitative PCR primer, kit and detection method for detecting porcine circovirus 3
  • Real-time fluorescent quantitative PCR primer, kit and detection method for detecting porcine circovirus 3

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Embodiment 1

[0042] 1. Primer design and synthesis

[0043] According to the published PCV3 gene sequence, utilize Primer 6.0 software to design a pair of specific fluorescent quantitative PCR primers F1 and R1 according to the conserved sequence of PCV3 ORF2 gene, the amplification length is 151bp; The fluorescent quantitative PCR primer sequence is:

[0044] Upstream primer F1: 5'-TTTCCGGGACATAAATGCT-3';

[0045] Downstream primer R1: 5'-TACTTCACCCCCAAACCAA-3'.

[0046] Design a pair of primers Q1 and Q2 to amplify the whole gene of PCV3 ORF2, the length of the primers is 645bp; the primer sequence of the whole gene of PCV3 ORF2 is:

[0047] Upstream primer Q1: 5'-TTAGAGAACGGACTTGTAACGAATC-3';

[0048] Downstream primer Q2: 5'-ATGAGACACAGAGCTATATACAGAA-3'.

[0049] 2. Preparation of recombinant plasmid standard

[0050] (1) Extraction of viral nucleic acid

[0051] PCV3-positive disease tissue (from a pig farm in Guangdong, identified as PCV3-positive according to the PCV3 standard ...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a real-time fluorescent quantitative PCR primer, kit and detection method for detecting the porcine circovirus 3. The nucleotide sequence of the real-time fluorescent quantitative PCR primer for detecting the porcine circovirus 3 is shown in SEQ ID NO.1-2. The invention further provides the kit containing the primer mentioned above and the detection method for detecting the porcine circovirus 3. The primer, the kit and the detection method are high in specificity, high in sensitivity and stable in repeatability, wave crests of the melting curve of amplified products are single, no primer dimer is caused, and the primer and the kit have no cross reaction with genomes of the porcine circovirus 2, the hog cholera virus, the porcine pseudorabies virus and the porcine reproductive and respiratory syndrome virus; sensitivity is 102 copies per microliter and is 100 times higher than that of the conventional PCR; and the variation coefficients of inter-group and intra-group repeated tests are all smaller than 3.0%. The foundation is laid for research on molecular epidemiology, prevention and control of the pestilence.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time fluorescent quantitative PCR primer, a kit and a detection method for detecting porcine circovirus type 3. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) was first isolated in 1974 from the pig kidney cell line PK-15 by German scholar Tischer et al. PCV is the smallest animal virus found so far. The diameter of the virus particle is about 17nm, and it is icosahedral. According to the sequence of discovery, pathogenicity and genome sequence, there are currently three types of circoviruses, namely PCV1 (Porcine circovirus 1), PCV2 (Porcine circovirus 2) and PCV3 (Porcine circovirus 3). PCV1 was first found to be non-pathogenic to pigs. The subsequently discovered PCV that can cause disease in pigs is called PCV2. PCV2 has been shown to be the main causative agent of diseases such as postweaning mμLtisystemic wasting s...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 樊惠英谢永生王诚诚
Owner SOUTH CHINA AGRI UNIV
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