Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs
Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs
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DOI：
10.1016/j.jviromet.2005.10.021
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发表时间：
2006-04-01
影响因子：
3.1
通讯作者：
Park, Bong-Kyun
中科院分区：
文献类型：
--
作者：
Song, Dae-Sub;Kang, Bo-Kyu;Park, Bong-Kyun
猪流行性腹泻病毒（PEDV）是冠状病毒科的一种，在韩国养猪业中引起了毁灭性的肠道疾病。以前，使用限制性片段长度多态性分析（RFLP）确定了强毒田间PEDV毒株和Vero细胞培养适应的PEDV DR 13毒株之间的差异，并报道了猪中的PEDV散毒模式。在这些研究的扩展中，构建了内部对照，并建立了经口接种后病毒脱落的定量分析。将亲本田间PEDV和细胞培养物适应的PEDV DR 13口服接种至缺乏初乳的1日龄仔猪、商业2周龄猪和母猪（1-5 ml剂量，10（5.8）× 10（6.0）TCID 50/0.1ml）。每天监测PEDV脱落，并使用定量逆转录酶聚合酶链反应（RT-PCR）方法测量病毒水平。在实验接种猪的粪便样品中，排泄的病毒水平在口服接种后2天达到峰值，此后逐渐下降。此外，使用相同的RT-PCR方法对来自田间样品的PEDV进行定量以确定脱落病毒载量，这表明通过定量RT-PCR测量猪中PEDV脱落病毒载量可能是评估PEDV在猪群体中传播潜力的有用工具。(c)2005 Elsevier B. V.保留所有权利。
Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined Using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1-5 ml dose, 10(5.8)10(6.0) TCID50/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load.This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population. (c) 2005 Elsevier B.V. All rights reserved.