Huang Yong, Xing Na, Wang Zengguo, Zhang Xiujuan, Zhao Xiaomin, Du Qian, Chang Lingling, Tong Dewen
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China.
PLoS One. 2015 Nov 6;10(11):e0141545. doi: 10.1371/journal.pone.0141545. eCollection 2015.
Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.
在现代集约化养猪生产中，多种病毒的混合感染很常见。然而，在临床前水平上，尚无方法可在同一反应体系中同时检测DNA病毒和RNA病毒。在本研究中，我们旨在开发一种基于双链超灵敏纳米颗粒DNA探针的PCR检测方法（双链UNDP-PCR），该方法能够在同一反应体系中同时检测DNA病毒和RNA病毒。选择猪圆环病毒2型（PCV2）和猪传染性胃肠炎病毒（TGEV）作为两种不同类型病毒的代表。通过与裂解缓冲液一起煮沸，从血清样本中同时释放出PCV2 DNA和TGEV RNA，然后加入包被有针对TGEV和PCV2的单链和/或双链特异性探针的磁珠和金纳米颗粒，与病毒释放的核酸形成类似夹心的复合物。磁分离后，使用二硫苏糖醇（DTT）洗脱PCV2和TGEV特异性的DNA条形码，随后通过针对特异性DNA条形码的特异性PCR检测进行鉴定。双链UNDP-PCR显示出与单链UNDP-PCR相似的灵敏度，能够检测血清中PCV2和TGEV各20个拷贝，比传统的双链PCR/RT-PCR检测方法灵敏度高约250倍。未观察到与其他病毒的交叉反应。基于单链MMPs和双链MMPs 的双链UNDP-PCR的阳性检出率相同，PCV2为29.6%，TGEV为9.3%，PCV2和TGEV混合感染为3.7%。这种双链UNDP-PCR检测方法无需进行DNA/RNA提取、纯化以及RNA逆转录，即可同时从大规模血清样本中检测TGEV（RNA病毒）和PCV2（DNA病毒），并且对于PCV2（29%）和TGEV（11.7%）临床前感染的阳性检出率比传统双链PCR/RT-PCR显著提高。因此，所建立的双链UNDP-PCR是一种快速且经济的检测方法，具有高灵敏度、特异性和可重复性。
