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Method, primer, probe and kit for detecting porcine circovirus through triple digital microdroplet PCR

A porcine circovirus and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of large interference, easy false negative results, complex composition and matrix, etc.

Pending Publication Date: 2021-08-31
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Claims
  • Application Information

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Problems solved by technology

The qPCR method has certain advantages in the detection of routine samples such as animal tissues, but animal-derived products, especially deep-processed products such as feed and meat products, often have complex composition matrices and contain a large number of inhibitors, which makes conventional methods such as qPCR more susceptible to interference. Large, prone to false negative results

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  • Method, primer, probe and kit for detecting porcine circovirus through triple digital microdroplet PCR
  • Method, primer, probe and kit for detecting porcine circovirus through triple digital microdroplet PCR
  • Method, primer, probe and kit for detecting porcine circovirus through triple digital microdroplet PCR

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Embodiment Construction

[0062] 1. Design and synthesis of primers and probes

[0063] According to the PCV1 gene sequence (KC447455.1), PCV2 gene sequence (MG245866) and PCV3 gene sequence (NC_031753.1) registered in GenBank as templates, Primer3 was used to design specific primers and specific TaqMan for PCV1, PCV2 and PCV3 respectively Probes, wherein the probe P3 of PCV3 uses HEX as the fluorescent luminescent group, the PCV2 probe is labeled with the FAM fluorescent group, and the PCV1 probe P1 uses Cy5 as the luminescent group (Table 1). And design plasmid standards according to each genome sequence. Primers and probes were synthesized by TAKARA Company.

[0064] Table 1 Amplification primers and probe sequences

[0065]

[0066] 2. Establishment of dPCR detection method

[0067] 2.1 dPCR workflow

[0068] Using Naica TM The CrystalDigitalPCR system carries out CrystaldPCR (hereinafter referred to as cdPCR) amplification. The experimental operation steps include preparation of PCR reacti...

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Abstract

The invention relates to the field of epidemic prevention detection, in particular to a method, primer, probe and kit for detecting porcine circovirus through triple digital microdroplet PCR. According to an application of a detection target spot to detection of the porcine circovirus, the detection is a non-disease detection and diagnosis method, and the detection target spot comprises one or more of the following detection target spots: the sequence of the porcine circovirus type 1 (PCV1) detection target spot is shown as SEQ ID NO: 1; the sequence of a porcine circovirus type 2 (PCV2) detection target is as shown in SEQ ID NO: 2; and the sequence of a porcine circovirus type 3 (PCV3) detection target is as shown in SEQ ID NO: 3. Based on the advantages of ddPCR, a high-sensitivity and accurate identification and detection method for different genotypes of the porcine circovirus is established, and the method can be applied to conventional animal quarantine and quarantine monitoring and load titration of animal-derived products with low virus content and complex matrixes such as feeds, meat products and vaccines.

Description

technical field [0001] The invention relates to the field of epidemic prevention detection, in particular to a method for detecting porcine circovirus by triple digital droplet PCR, primers, probes and a kit. Background technique [0002] According to the latest "Terrestrial Animal Health Code" released by the OIE, animal-derived deep-processed products such as feed and meat products are defined as important risk points for the spread of animal diseases. Porcine circovirus, as a second-class infectious disease, is mainly divided into porcine circovirus type 1, porcine circovirus type 2, and porcine circovirus type 3, which are widely spread in pig farms all over the world. Due to the wide consumption demand of pork and its products in my country, coupled with the high temperature and acid resistance of PCV2 itself, pig-derived products have become an important biosafety risk point for the spread of PCV2. [0003] Real-time fluorescent quantitative PCR (Real-time fluorescent...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2563/159C12Q2537/143C12Q2563/107
Inventor 张晓峰帅江冰金晨晨曾若雪
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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