Development of rea1-time fluorescent quantitative PCR assay based on TaqMan probe for detection of porcine parvovirus

The structural protein 2(VP2) nucleotide sequences of porcine parvovirus(PPV) were retrieved from the GenBank and aligned using the software program DNAstar.One pair of primers and a TaqMan probe were designed using the Primer express(version 2.0) software based on a highly conserved sequence within the VP2 region of the PPV genome.VP2 gene was amplified by PCR from DNA extracted from the PK-15 porcine kidney cells infected by 7909 isolates of PPV,and then was cloned and sequenced.And the positive recombination plasmid was used as a positive quantitative template to establish a standard curve.In a 25 μL reaction,real-time fluorescent quantitative PCR was established by optimizing the probe's concentration,Mg2+concentration,primers concentration and the annealing temperature,rea1-time fluorescent quantitative PCR was established.When the specificity of the assay using the specific PPV primer was evaluated by testing the PPV standard strain and other viruses,no cross-reactions were detected with non-PPV reference viruses.Detection limit of real-time PCR for PPV was 1.12 TCID50/mL.A real-time PCR assay was performed on 80 clinical samples and compared with conventional PCR assay.In 48 of 80 samples,PPV DNA was detected by the conventional PCR assay.All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time assay,and 12 of 32 samples that tested negative for PPV DNA by the conventional assay tested positive by the real-time PCR assay.In real-time PCR assay,the number of PPV was detected increased by 15%.Therefore,it is considered to be a useful tool for the detection of PPV.