The Establishment Of A Rapid Detection Method Of PCV2Using An Ultrasensitive Nanoparticle And DNA Probe-based PCR Assay

Porcine circovirus type2(PCV2) has emerged as one of the most importantpathogens affecting swine production globally. Preclinical identification of PCV2isvery important for effective prophylaxis of PCV2-associated diseases. In this study, wedeveloped an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR)for PCV2detection. Magnetic microparticles coated with PCV2specific DNA probeswere used to enrich PCV2DNA from samples, then gold nanoparticles coated withPCV2specific oligonucleotides were added to form a sandwich nucleic acid-complex.After the complex was formed, the oligonucleotides were released and characterizedby PCR. Firstly, to expand the breadth of UNDP-PCR assay as much as possible, weselected the highly conserved sequence of PCV2as targeted regions for designingprobes1-8. The designed probes were coated to MMP, respectively, to preparefunctionalized magnetic microparticles. The optimal probes for capture of PCV2genomic DNA were selected and determined by testing the capture efficiency of theseMMPs for genomic DNAs from different serotypes and genotypes of PCV2strains.Next, we evaluated the conjugation and amplification efficiency of functionalizedAu-NPs with different oligonucleotides in UNDP-PCR assay. At last,we verified thesensitivity, specificity, and reproducibility of the UNDP-PCR assay. From theexperiments, the following results were obtained:1. We selected the highly conserved sequence of length18-35bp in the capsidprotein-coding region from about35different PCV2a, PCV2b and recombinationstrains, and aligned with PCV1to exclude the regions with higher homology withPCV1.Then, we determined8targeted regions for designing probes. The designedprobes1-8were coated to MMP, respectively, to prepare functionalized magneticmicroparticles MMP-p1,-p2,-p3,-p4,-p5,-p6,-p7and-p8. The optimal probes forcapture of PCV2genomic DNA were selected and determined by testing the captureefficiency of these MMPs for genomic DNAs from different serotypes and genotypes of PCV2strains. In these probes-functionalized MMPs, probe5-functionalizedmagnetic microparticles are optimal for preconcentration of various PCV2strainsDNA and separation of sandwich nucleic acid-complex in this assay. oligo4-functionalized Au-NPs is optimal for binding and amplification of detected DNAsignals in this assay. Ultimately, we developed an ultrasensitive nanoparticle DNAprobe-based PCR assay (UNDP-PCR) for PCV2detection.2. This assay exhibited about500-fold more sensitive than conventional PCR,with a detection limit of2copies of purified PCV2genomic DNA and10viral copiesof PCV2in serum. The assay has a wide detection range for all of PCV2genotypeswith reliable reproducibility. No cross-reactivity was observed from the samples ofother related viruses including porcine circovirus type1, porcine parvovirus, porcinepseudorabies virus, porcine reproductive and respiratory syndrome virus and classicalswine fever virus.3. The positive detection rate of PCV2specific UNDP-PCR in40subclinical fieldsamples was27.5%, which appeared greater than that by conventional and real-timePCR and appeared application potency in evaluation of the viral loads levels ofpreclinical infection samples. The UNDP-PCR assay reported here can reliably rule outfalse negative results from antibody-based assays, provide a nucleic acid extractionfree, specific, ultrasensitive, economic and rapid diagnosis method for preclinicalPCV2infection in field, which may help prevent large-scale outbreaks.