Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.
Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.
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DOI：
10.1371/journal.pone.0141545
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Tong D
中科院分区：
文献类型：
--
作者：
Huang Y;Xing N;Wang Z;Zhang X;Zhao X;Du Q;Chang L;Tong D
多种病毒的混合感染在现代集约化养猪中很常见。然而，在临床前水平上，没有方法可以在同一反应体系中检测DNA和RNA病毒。在这项研究中，我们的目的是开发一种双重超灵敏的纳米粒子DNA探针为基础的PCR检测（双重UNDP-PCR），能够同时检测DNA和RNA病毒在同一个反应系统。选择PCV 2和TGEV作为两种不同类型病毒的代表。用裂解缓冲液煮沸，同时从血清样品中释放出PCV 2 DNA和TGEV RNA，然后加入磁珠和包被有TGEV和PCV 2的单和/或双链特异性探针的金纳米颗粒，与病毒释放出的核酸形成类DNA复合物。在磁性分离之后，使用DTT洗脱对PCV 2和TGEV具有特异性的DNA条形码，随后通过特异性PCR测定对特异性DNA条形码进行表征。双重UNDP-PCR显示出与单一UNDP-PCR相似的灵敏度，并且能够在血清中检测到PCV 2和TGEV各20个拷贝，显示出比常规双重PCR/RT-PCR测定高约250倍的灵敏度。与其他病毒无交叉反应。以MMPs为基础的双重UNDP-PCR和单一MMPs为基础的双重UNDP-PCR的阳性检出率相同，PCV 2为29.6%，TGEV为9.3%，PCV 2和TGEV混合感染为3.7%。该双重UNDP-PCR方法可同时检测大规模血清样本中的TGEV（RNA病毒）和PCV 2（DNA病毒），无需DNA/RNA提取、纯化和RNA逆转录，对PCV 2和TGEV临床前感染的阳性检出率（29%和11.7%）较常规双重PCR/RT-PCR显著提高。因此，建立的双重UNDP-PCR方法是一种快速、经济的检测方法，具有较高的灵敏度、特异性和重复性。
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.