Huang Yong, Zhang Xiujuan, Du Qian, Wang Fengyu, Zhao Xiaomin, Zhang Wenlong, Tong Dewen
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P. R. China.
PLoS One. 2014 May 19;9(5):e97869. doi: 10.1371/journal.pone.0097869. eCollection 2014.
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.
猪圆环病毒2型（PCV2）已成为影响全球养猪业的最重要病原体之一。PCV2的临床前鉴定对于有效预防PCV2相关疾病非常重要。在本研究中，我们开发了一种基于超灵敏纳米颗粒DNA探针的PCR检测方法（UNDP-PCR）用于PCV2检测。用PCV2特异性DNA探针包被的磁性微粒用于从样品中富集PCV2 DNA，然后加入用PCV2特异性寡核苷酸包被的金纳米颗粒形成夹心核酸复合物。复合物形成后，释放寡核苷酸并通过PCR进行鉴定。该检测方法的灵敏度比传统PCR高约500倍，纯化的PCV2基因组DNA的检测限为2拷贝，血清中PCV2的病毒拷贝数检测限为10个。该检测方法对所有PCV2基因型具有广泛的检测范围，具有可靠的重复性。在包括猪圆环病毒1型、猪细小病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒和经典猪瘟病毒在内的其他相关病毒样品中未观察到交叉反应。40份临床前现场样品中PCV2特异性UNDP-PCR的阳性检出率为27.5%，高于传统PCR和实时PCR，在评估临床前感染样品的病毒载量水平方面具有应用潜力。本文报道的UNDP-PCR检测方法可以可靠地排除基于抗体检测的假阴性结果，为现场临床前PCV2感染提供一种无需核酸提取、特异性强、超灵敏、经济且快速的诊断方法，这可能有助于预防大规模疫情爆发。
