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Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus

A technology of porcine pseudorabies virus and porcine circovirus, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as hazards and cross-contamination, and achieve good application stability and low cost.

Inactive Publication Date: 2010-09-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are many methods for detecting various viruses. For example, the methods for detecting latent PRV infection include serological tests, activation and detection of latent viruses in vivo, tissue block culture and co-cultivation techniques, nucleic acid hybridization and conventional PCR detection techniques, of which conventional PCR detection The technical sensitivity, specificity, stability and operability are relatively good, but the treatment after the conventional PCR reaction has cross-contamination and EB hazards to the operator

Method used

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  • Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus
  • Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus
  • Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Standard plasmid preparation

[0040] 1 Preparation of target fragments Extract nucleic acids of PRV, PPV, PCV-2, and HCV according to conventional methods, and store the nucleic acids at -20°C for later use. To design primers, use OLIGO6.24, compare the nucleic acid sequences in GENEBANK, and design primers (Table 1). The extracted PRV, PPV, PCV-2, and HCV viral nucleic acids were used as templates, and the primers in Table 1 were used for PCR amplification. The length of the amplified products was expected to be 265bp, 764bp, 792bp, and 303bp. The amplified products obtained were subjected to agarose gel electrophoresis, and the results were shown in figure 1 .

[0041] Table 1 Sequences of primer pairs for amplifying target genes

[0042]

[0043] 2 Preparation of standard plasmids Recover the target fragments according to the Plasmid Miniprep Kit operation method, connect the recovered products to the pMD18-T vector, transform Escherichia coli DH5α ...

Embodiment 2

[0045] Example 2 Establishment of SyMuReTi-PCR detection method for 4 kinds of viruses

[0046] 1.1 The dilution of the four virus standard plasmids is 4.97×10 using the Eppendorf Biophotometer to quantify the standard plasmids 12 Copies / μl, 3.44×10 12 Copies / μl, 8.18×10 12 Copies / μl, 7.98×10 12 Copies / μl, use EASY Dilution to dilute the standard plasmid at 1.0×10 8 ~1.0×10 1 Copies / μl 10-fold dilution, stored at -20°C.

[0047] 1.2 Detection primer design The target gene cloned in Example 1 was sequenced, and according to the determined sequence, four pairs of specific primers were designed using OLIGO6.24 (Table 2).

[0048] Table 2 Sequences of primer pairs for amplifying target genes

[0049]

[0050] 1.3 Add SyMuReTi-PCR to an Axygen Scientific Quality centrifuge tube (200 μl) in sequence: 9.5 μl of sterilized deionized water, 1 μl of dimethyl sulfoxide, 0.5 μl of each of the 4 pairs of primers in Table 2, and add different dilutions to different centrifuge tubes...

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Abstract

The invention discloses a multiplex real-time fluorescent quantitation PCR method capable of detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and hog cholera virus at the same time. The method of the invention detects that the four virus are all in good linear relations at the same time, all ten times serial dilution points of constructed normal plasmid are all in one straight line, CT value and copy number are in good linear relation, and regression analysis shows that the related coefficient of the CT value and the copy number is R<2> more than 0.99.The multiplexreal-time fluorescent quantitation PCR method has the advantages of excellent specificity, sensibility and stability, which can rapidly, sensitively and differentially detect the four virus with serious harm to the economy and can be used for early diagnosis of virus infection.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a multiple real-time fluorescence quantitative PCR method for simultaneous detection of porcine circovirus, porcine parvovirus, porcine pseudorabies virus and swine fever virus, belonging to the field of biotechnology. Background technique [0002] Porcine pseudorabies virus (Porcine pseudorabies virus, PRV), porcine parvovirus (Porcine parvovirus, PPV), type II porcine circovirus (Porcine circoviruses, PCV2), swine fever virus (Hog cholesterolavirus, HCV) etc. all belong to porcine respiratory diseases and The pathogen of reproductive disorder syndrome, and the mixed infection of 4 viruses often occurs. PCV-2 is also associated with porcine respiratory disease complex (PRDC), postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). ) etc. are closely related and are important primary pathogens causing various syndromes (Segales J, Alla...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李曦童光志符芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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