Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus.
Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus.
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DOI：
10.1016/j.jviromet.2007.06.021
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发表时间：
2007-12
影响因子：
3.1
通讯作者：
Hyun BH
中科院分区：
文献类型：
--
作者：
Kim SH;Kim IJ;Pyo HM;Tark DS;Song JY;Hyun BH
猪传染性胃肠炎病毒（TGEV）和猪流行性腹泻病毒（PEDV）是引起仔猪腹泻和死亡的主要病原。多重实时逆转录酶（RT）-PCR被开发用于在单个反应管中使用基于来自TGEV和PEDV核衣壳基因的序列的Cy 5-和FAM-标记的TaqMan-探针同时差异定量每种病毒。使用该测定分别在9 × 107至9 × 101拷贝和7 × 107至7 × 101拷贝的范围内定量TGEV和PEDV转录本的拷贝数。使用每种转录物的标准品溶液评价试验内和试验间的变异性，变异系数（CV）分别小于3.43%和3.33%。用强毒TGEV和PEDV实验感染仔猪，并测量从腹泻开始的病毒量。从经历PED或TGE的农场获得的样品定量为102至105个RNA拷贝。总之，该检测方法为检测和定量病毒载量提供了有效的病原学诊断工具。该检测方法也可能被证明用于检测感染，最终导致更好的农场疾病控制。
Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are major etiological agents of diarrhea and death in piglets. Multiplex real-time reverse transcriptase (RT)-PCR was developed for simultaneous differential quantification of each virus in a single reaction tube, using Cy5- and FAM-labeled TaqMan-probes based on sequences from the TGEV and PEDV nucleocapsid genes. The copy numbers for transcripts of TGEV and PEDV were quantified using this assay over a range from 9 × 107 to 9 × 101 copies and 7 × 107 to 7 × 101 copies, respectively. The variability of the intra-assay and inter-assay were evaluated using standard solutions of each transcript, with coefficients of variation (CV) less than 3.43 and 3.33%, respectively. Piglets were experimentally infected with virulent TGEV and PEDV, and the amounts of virus from the onset of diarrhea were measured. Samples obtained from farms experiencing PED or TGE were quantified between 102 and 105 RNA copies. In conclusion, this assay provides an effective etiological diagnostic tool for detecting and quantifying viral loads. The assay may also prove useful for detecting infections, ultimately leading to better disease control on farms.
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