Establishment and application of PCR assay for detect of pigeon circovirus
To establish a method for the rapid and sensitive detection of pigeon circovirus(PiCV),a genome-wide recombinant plasmid of PiCV had been constructed according to a PiCV popular strain genome sequence(GenBank:OL901205.1).Subsequently,multiple sets of primers and probes were designed based on the conserved sequence of PiCV Rep gene.After screening,the optimized primers and probes com-bination which presented the best amplification efficiency was selected.And the TaqMan fluorescent quantitative PCR detection method was established.To test the sensitivity of this method,the synthet-ic standard recombinant plasmid was employed.The results showed that the minimum detection limit of template is 21 copies/μL and the data can keep a good linear relationship over the range 2.1 × 101-2.1 × 108 copies/μL with the linear correlation coefficient R2=0.999 1.This method also show a highly specificity to PiCV which has no signal can be detected from pigeon herpesvirus type 1,pigeon aden-ovirus,Newcastle disease virus and pigeon rotavirus.The repeatability of the method is also excel-lent.The coefficient of variation is less than 2％,and the intra-assay coefficient of variation is less than l％.By using 50 clinical samples,the positive detection rate of this method is 94％while that of PCR is 82％.The coincidence rate of the two methods was 100％.In summary,the TaqMan fluorescent quantitative PCR detection method of PiCV established in this experiment can be used for clinical de-tection of PiCV.
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