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Real-time fluorescent quantitative PCR-HRM primers for detecting PCV1 and PCV3

A PCR-HRM, real-time fluorescence quantitative technology, applied in the field of animal epidemiology, can solve problems such as difficult to achieve research goals, high possibility of crossover, tedious and complicated optimization of primer conditions, etc.

Inactive Publication Date: 2019-05-10
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, the condition optimization of the 4 primers is cumbersome and complicated, and the more primers, the greater the possibility of potential crossover, making it difficult to achieve relevant research purposes

Method used

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  • Real-time fluorescent quantitative PCR-HRM primers for detecting PCV1 and PCV3
  • Real-time fluorescent quantitative PCR-HRM primers for detecting PCV1 and PCV3
  • Real-time fluorescent quantitative PCR-HRM primers for detecting PCV1 and PCV3

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Embodiment 1

[0024] 1. Virus strain:

[0025] Porcine circovirus type 1 (PCV1) and porcine circovirus 3 (PCV3), porcine parvovirus (PPV), classic porcine pseudorabies virus (PRV), variant porcine pseudorabies virus (V-PRV), pig reproduction and Respiratory Syndrome Virus (PRRSV) and Porcine Epidemic Diarrhea Virus (PEDV) were isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0026] 2. Primer design and synthesis

[0027] 2.1 PCV1 and PCV3 genome sequence alignment

[0028] Select the representative strain of PCV1 (PK strain, GenBank accession number DG650650; BJ-1 strain, GenBank accession number FJ475129) PCV3 representative strain (CN2018LN-2 strain, GenBank accession number MH277116; CN2018LN-3 strain, GenBank accession number MH277117) as an example, The DNAStar biology software was selected for nucleotide homology comparison, and the results showed that: the nucleotide homology between PCV1 is over 99.7...

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Abstract

The invention relates to a group of real-time fluorescent quantitative PCR-HRM primers for detecting the porcine circovirus type 1 and the porcine circovirus type 3, and belongs to the field of animalinfectious disease. The primers are designed according to nucleotide-sequence characteristic differences of the PCV1 and PCV3 gene group in rep protein (Rep), obvious characteristic nucleotide sequence differences (differences exist in the GC% content) exist in the PCV1 and the PCV3 of a difference area, thus, melting temperature (Tm) peak value differences of the PCV1 and the PCV3 exist in the area, and after amplification is conducted with the established real-time fluorescent quantitative PCR method, different Tm peak values can appear, and the PCV1 and the PCV3 are distinguished. The PCV1and the PCV3 can be distinguished and diagnosed only through a group of primers, and meanwhile the inflection degree when pigs inflect the PCV1 and the PCV3 can be specifically made clear. The application method is simple and effective, the cost is low, and the accuracy is high.

Description

Technical field [0001] The present invention relates to a set of real-time fluorescent quantitative PCR-HRM primers for detecting porcine circovirus type 1 and type 3 (PCV1 and PCV3), and belongs to the field of animal infectious diseases. Background technique [0002] High Resolution Melt (HRM) is a simple, rapid, and low-cost PCR amplification detection technology that can be used for high-throughput mutation scanning and genotyping. This technology can be carried out only by adding double-stranded DNA binding dye on the basis of standard PCR reagents, without sequence-specific probes, and directly run high-resolution melting curves after PCR to complete the analysis of sample genotypes . When using SYBR Green I as a fluorescent dye, a one-step melting curve reaction program is usually run after real-time PCR amplification is completed. That is, the temperature of the PCR product is gradually increased from low to high. During the heating process, the double-stranded DNA is m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 陈如敬陈秋勇吴学敏车勇良王晨燕周伦江侯博严山王隆柏魏宏
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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