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Primers, probes and detection kits for detection of porcine circovirus, porcine pseudorabies virus and porcine parvovirus

A technology for porcine pseudorabies virus and porcine circovirus, which is applied in biochemical equipment and methods, microbial determination/inspection, material excitation analysis, etc.

Active Publication Date: 2011-12-14
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional single-item PCR and real-time quantitative PCR can only detect one virus at a time, which is time-consuming and labor-intensive. It is difficult to apply conventional methods for early and rapid detection when multiple causes of infection are mixed.

Method used

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  • Primers, probes and detection kits for detection of porcine circovirus, porcine pseudorabies virus and porcine parvovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1, the composition of kit

[0068] 1. The composition of the kit

[0069] 1) DNA extraction reagent; DNA extraction kit (Genomic DNA Purification kit, cat.no K201) of Shanghai Bocai Biotechnology Co., Ltd.;

[0070] 2) DNA standard product: T vector plasmid containing porcine circovirus target gene, T vector plasmid containing porcine pseudorabies virus target gene and T vector plasmid containing porcine parvovirus target gene;

[0071] 3) Real-time quantitative PCR reaction solution, which includes: 1 × PCR buffer containing Taq DNA polymerase and dNTP, 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for detecting porcine circovirus, for detecting porcine circovirus 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for pseudorabies virus, 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for detecting porcine parvovirus, 1× fluorescent quantitative PCR reference dye;

[0072] Furthe...

Embodiment 2

[0083] Embodiment 2 utilizes DNA standard substance to establish standard curve

[0084] The concentration of the above-mentioned plasmid DNA (PCV-T, PRV-T, PPV-T) that has been measured, using the formula: (6.02 x 10 23 copy number / mole) x (concentration g / ml) x10 -3 / (MW g / mol) = copy / μl, respectively calculate PCV-T, PRV-T and PPV-T copy number, common ten-fold dilution, with 10 3 ~10 7The copy / μl RNA is used as the template, and the reaction is carried out on the ABI7300 fluorescent quantitative PCR instrument. The reaction system is 20 μl, of which 2 x premix Ex TaqTM 10 μl, PCV(F) and PCV(R), PPV(F) and PPV(R) 0.2μl each of PRV(F) and PRV(R) (20μM), 0.4μl each of PCV-Probe, PPV-Probe and PRV-Probe (20μM), 0.4μl of ROX Reference Dye (50x), 2μl of template DNA, DDW to 20 μl. The reaction conditions were 95°C for 10 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The data was analyzed by software to obtain a standard curve. Their R valu...

Embodiment 3

[0085] Embodiment 3, the specificity test of kit

[0086] 1 Materials, see Table 2:

[0087] Virus source porcine respiratory and reproductive disorder syndrome virus Shanghai Haili Biological Pharmaceutical Co., Ltd. porcine circovirus Separation, identification and preservation of pig disease group of Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences porcine pseudorabies virus Wuhan Keqian Animal Biological Products Co., Ltd. porcine parvovirus Bought from China Veterinary Drug Control Institute swine fever virus Nanjing Tianbang swine fever cell vaccine porcine encephalitis virus Hunan Yahua Seed Industry Co., Ltd. Biopharmaceutical Factory porcine rotavirus Separation, identification and preservation of pig disease group of Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences Porcine E. coli Separation, identification ...

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Abstract

The invention discloses a primer, probe and assay kit used for detecting porcine circovirus, porcine pseudorabies virus and porcine parvovirus. The primer and probe used for detecting are shown as sequences from SEQIDNO:1 to SEQIDNO:9 in a sequence table. The invention also provides an assay kit used for detecting the porcine circovirus, the porcine pseudorabies virus and porcine parvovirus. The primer and the probe which are adopted by the invention have strong specificity, and three viruses can be detected in one step, thus cost is saved, steps are reduced, and efficiency is improved. The method provided by the invention has the characteristics of high specificity, high sensitivity, high efficiency and low cost and is applicable to analysis of a larger number of samples.

Description

technical field [0001] The invention relates to primers, probes and detection kits for detection of porcine circovirus, porcine pseudorabies virus and porcine parvovirus. Background technique [0002] Porcine circovirus type 2 (PCV-2), porcine pseudorabies virus (PRV) and porcine parvovirus (Porcine parvovirus, PPV) are currently common DNA viruses that cause reproductive disorders in pigs. At present, many pig farms have diseases caused by the above three viruses at the same time, causing huge economic losses to the pig industry. The above three viruses often show mixed infection. Clinically, the main characteristics of infected sows are stillbirth, deformed fetus, mummified fetus and sick piglets, which are not easy to distinguish. [0003] The current commonly used diagnostic techniques for swine diseases at home and abroad mainly include: clinical diagnosis, pathogenic diagnosis, serological diagnosis, molecular biological diagnosis, etc. Clinical diagnosis is mainly b...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 王一成袁秀芳徐丽华李军星郭勇
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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