Laboratory for Diagnosis of Porcine and Bovine Diseases, Federal Centre for Animal Health, FGI ARRIAH, Yur'evets, Vladimir 600901, Russia.
J Virol Methods. 2010 May;165(2):283-93. doi: 10.1016/j.jviromet.2010.02.010. Epub 2010 Feb 17.
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in the amplification step two different amplicons with sizes of 950 bp and 317 bp and (2) three pairs of internal primers in a second round of PCR (nested PCR), yielding two different amplicons with sizes of 792 bp and 208 bp for TGEV and porcine PRV-A, respectively. The genome of PEDV was not detected after the amplification step but it was detected in the second round of PCR, yielding amplicon with size of 291 bp. Multiplex nested RT-PCR can detect TGEV, PRV-A, and PEDV up to concentration 10(2) TCID(50)/mL, 10(1) TCID(50)/mL, and 27.2 microg/microl of RNA, respectively. A total of 175 field samples were collected from swine with diarrhea from January 2005 until July 2007. The samples were tested for the presence of three viruses by a multiplex nested RT-PCR. Dual infections with PEDV and PRV-A were identified in seven specimens (4%) (n = 6). Twenty-one (25%) infections were caused by PEDV and thirty-four infections (41%) were caused by PRV-A. The genome of TGEV was not detected in any of these field samples, however TGEV was detected in piglets infected experimentally. The multiplex nested RT-PCR is rapid, sensitive, and a cost-effective detection method for the detection of porcine enteric viruses.
猪流行性腹泻病毒（PEDV）、传染性胃肠炎病毒（TGEV）和猪 A 群轮状病毒（PRV-A）是引起仔猪肠病的主要病毒。本研究建立了一种用于检测仔猪腹泻病野外样本中这些病毒的多重巢式逆转录聚合酶链反应（multiplex nested RT-PCR）。该方法由（1）三组外部引物组成，在扩增步骤中产生两个大小分别为 950 bp 和 317 bp 的不同扩增子，以及（2）第二轮 PCR（巢式 PCR）中的三组内部引物组成，产生两个大小分别为 792 bp 和 208 bp 的不同扩增子，用于检测 TGEV 和猪 PRV-A。PEDV 基因组在扩增步骤后未被检测到，但在第二轮 PCR 中被检测到，产生大小为 291 bp 的扩增子。多重巢式 RT-PCR 可检测到浓度为 10(2)TCID(50)/mL、10(1)TCID(50)/mL 和 27.2 microg/microl 的 TGEV、PRV-A 和 PEDV 的 RNA。2005 年 1 月至 2007 年 7 月期间，从患有腹泻的猪群中采集了 175 份野外样本。采用多重巢式 RT-PCR 检测了这些样本中三种病毒的存在情况。在七个样本（4%）（n = 6）中发现了 PEDV 和 PRV-A 的双重感染。21 份（25%）感染是由 PEDV 引起的，34 份（41%）感染是由 PRV-A 引起的。在这些野外样本中均未检测到 TGEV 的基因组，但在感染实验的仔猪中检测到了 TGEV。该多重巢式 RT-PCR 是一种快速、敏感、经济有效的检测猪肠病毒的方法。
