Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.
Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of 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 virus, porcine reproductive and respiratory syndrome virus and classical swine fever 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 negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Yong
Y
Huang
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Xiujuan
X
Zhang
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Qian
Q
Du
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Fengyu
F
Wang
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Xiaomin
X
Zhao
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Wenlong
W
Zhang
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
Dewen
D
Tong
D000818:Animals; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D017925:Circovirus / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D058185:Magnetite Nanoparticles; D009841:Oligonucleotides / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000517:prevention & control / Q000821:virology*
D017931:DNA Primers; D015342:DNA Probes; D058185:Magnetite Nanoparticles; D009841:Oligonucleotides