College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
J Virol Methods. 2019 Jan;263:32-37. doi: 10.1016/j.jviromet.2018.10.009. Epub 2018 Oct 15.
Porcine viral diarrhea, mainly caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (RVA), porcine group C rotaviruses (RVC) and porcine circovirus 2 (PCV2), is a serious global problem, resulting in substantial economic losses to the swine industry. For fast and reliable diagnosis of the causative agent associated with viral diarrhea in pigs, an inexpensive and easy to perform gel-based multiplex PCR assay was developed in this study to detect and differentiate the different viruses by amplicon size. The assay was able to distinguish between all five viral agents without cross-reacting with other non-target pig viruses. The detection limits of the assay per reaction were 5 copies for PEDV, TGEV, RVC and PCV2 and 50 copies for RVA for the singleplex assays and 50 copies when all five viruses were multiplexed. Sixty-nine field samples were used to validate the developed multiplex assay. The overall prevalence of positive samples was 44.9% (31/69). PCV2 was detected in 37.7% of the samples, PEDV and RVC each in 4.3%, TGEV in 2.9%, and RVA was detected in 1.4% of the samples tested. A total of 5.8% of the samples were co-infected by two or more viruses, and the results of the multiplex assay were in agreement to those obtained by single PCR assays. These findings suggest that the developed cost-effective multiplex assay is specific, sensitive, and will serve as a valuable diagnostic tool for the rapid differential detection of these five viruses and for molecular epidemiological studies and diarrhea disease management.
猪病毒性腹泻主要由猪流行性腹泻病毒（PEDV）、传染性胃肠炎病毒（TGEV）、猪 A 群轮状病毒（RVA）、猪 C 群轮状病毒（RVC）和猪圆环病毒 2 型（PCV2）引起，是一个全球性的严重问题，给养猪业造成了巨大的经济损失。为了快速可靠地诊断引起猪病毒性腹泻的病原体，本研究开发了一种廉价且易于操作的凝胶基多重 PCR 检测方法，通过扩增子大小来检测和区分不同的病毒。该方法能够区分五种不同的病毒，且与其他非靶标猪病毒无交叉反应。该方法的检测限为：单重 PCR 检测时，PEDV、TGEV、RVC 和 PCV2 的检测限为 5 拷贝，RVA 的检测限为 50 拷贝；多重 PCR 检测时，五种病毒的检测限均为 50 拷贝。使用 69 份临床样本对该方法进行了验证。阳性样本的总检出率为 44.9%（31/69）。37.7%的样本中检测到 PCV2，PEDV 和 RVC 各占 4.3%，TGEV 占 2.9%，RVA 占 1.4%。共有 5.8%的样本同时感染两种或两种以上的病毒，且多重 PCR 检测结果与单重 PCR 检测结果一致。这些结果表明，该经济有效的多重 PCR 检测方法具有特异性、敏感性，可用于快速区分检测这五种病毒，以及进行分子流行病学研究和腹泻病管理。
