3 independent replicates assays of the UNDP-PCR showed highly consistent results (Fig. purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies computer virus, porcine reproductive and respiratory syndrome computer virus and classical swine fever computer virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false unfavorable results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and quick diagnosis method for preclinical PCV2 contamination in field, which may help prevent large-scale outbreaks. Introduction Porcine circovirus type 2 (PCV2) is the major etiological agent of porcine circovirus associated diseases (PCVAD), including postweaning multisystemic losing syndrome (PMWS), and porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), and congenital tremors type II (CT), which have caused heavy losses in global agriculture in recent years [1], [2], [3]. PCV2 serological studies showed that PCV2 contamination is usually ubiquitous all over the world, while prevalence of clinical disease is relative lower, suggesting that subclinical or preclinical contamination is the dominant form of PCV2 [4]. It has also been exhibited experimentally that subclinical PCV2 contamination may be associated with decreased vaccine efficacy [5]. Therefore, PCV2 subclinical contamination not only is the most common contamination form but also impact vaccine efficacy. Rapid and early identification of PCV2 subclinical contamination is very important for the effective prophylaxis of PCVAD. PCV2, belonging to the genus Circovirus of the family Pirozadil Circoviridae, are small nonenveloped DNA viruses containing a unique single-stranded circular genome of 1 1.7 kb [6]. The genomic DNA is usually packaged into a nonenveloped icosahedral capsid by capsid protein [7]. Antigenic studies have showed that PCV2 coat proteins possess six recognized linear epitopes [8]. Sequence alignments of field isolated PCV2 Pirozadil capsid proteins have recognized a number of variable regions corresponding to the recognized epitope sites [9],[10],[11]. Indeed, these studies have exhibited that antigenic differences in the capsid proteins exist among the different strains of PCV2 despite higher degree of sequence identity (90%) shared among their capsid proteins [12]. The antigenic difference exists among the different strains of PCV2 make it difficult to find an antibody that can be Pirozadil used to detect numerous PCV2 strains in field [13], but DNA probe targeted to the conserve sequence of different PCV2 strains can solve this problem. Therefore, to develop a DNA probe-based nanoparticle amplification method is very useful for detection of diverse PCV2 strains, especially for identification of PCV2 subclinical or preclinical contamination. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for preclinical identification of PCV2 contamination via systematical optimization. Magnetic microparticles (MMP) coated with optimal specific PCV2 DNA probes and platinum nanoparticles (AuNPs) coated with optimal specific PCV2 DNA probes and barcodes were used to enrich and amplify the poor signals from very small amount of PCV2 computer virus in serum samples. In each computer virus DNA-binding event, the platinum nanoparticles carry with it a large number of DNA barcodes, and subsequently release these DNA barcodes to be detected by PCR. Therefore, the nanoparticle DNA probe-based PCR can significantly enhance the sensitivity of standard PCR and to breakthrough the detection limit of standard PCR, to gain an innovative method suitable for preclinical diagnosis of PCV2 contamination, with greater sensitivity than the other conventional methods. Materials and Methods Materials, Reagents and Preclinical Samples Carboxylated-modified magnetic beads (MyOne Dynabeads; Invitrogen, Inc., Carlsbad, CA, USA) were utilized for preconcentration of PCV2 DNA and separation of sandwich nucleic Pirozadil acid-complex. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N-Hydroxysuccinimide were from Sigma-Aldrich (St. Louis, MO, USA) and utilized for activation of MyOne Dynabeads. DL-Dithiothreitol (DTT; Sigma-Aldrich, Inc., St. Louis, MO, USA) was utilized for the cleavage of oxidized thiolated oligonucleotides and release of thiolated barcode DNA from Au-NPs (Biopanda Reagents, Belfast, United Kingdom) surface. A total of 40 blood samples were collected from your Landrace pigs (ranging from 1 to 2 2 months of age) without apparently clinical symptoms. These blood samples were provided by 2 Pirozadil local farms (xianyang WNT16 & xingping) in the Shaanxi Province, China. The pigs were humanely euthanized with a dose of Ketamine (15 mg/kg. i.v. in the jugular venous external), then 5C10 ml of blood samples from each pig were collected by jugular venipuncture. The serum samples were tested using the commercial ELISA, PCR, real-time PCR and UNDP-PCR..
