Plus- and minus-stranded foot-and-mouth disease virus RNA quantified simultaneously using a novel real-time RT-PCR

文献类型： 外文期刊

第一作者： Gu, Chaojiang

作者： Gu, Chaojiang;Zheng, Congyi;Shi, Lili;Zhang, Qian;Li, Yong;Lu, Bin;Xiong, Yi;Qu, Sanpu;Shao, Junjun;Chang, Huiyun

作者机构：

关键词： FMDV;real-time RT-PCR;TaqMan probe;minus-strand;plus-strand;suckling mice;virions

期刊名称：VIRUS GENES （ 影响因子：2.332； 五年影响因子：2.0 ）

ISSN： 0920-8569

年卷期： 2007 年 34 卷 3 期

页码：

收录情况： SCI

摘要： Even though tagged RT-PCR and rTth RT-PCR have been developed to improve strand-specific detection of RNA virus, these assays are not quantitative. In this study, a novel real-time RT-PCR assay, which combines the benefits of both the tagged and rTth RT-PCR has been developed to quantify the strand-specific RNA of foot-and-mouth disease virus (FMDV). The tagged-primers plus a TaqMan probe located within the highly conserved viral 3D region were used. The in vitro synthesized minus-and plus-stranded RNA templates were used as a dual control along with the copy number of viral RNA molecules, which is more reliable than reported RT-PCR employing a DNA-based standard. This assay was used to quantify FMDV RNA from 10(9) to 10(1) copies with a maximum sensitivity of between ten and five copies and was shown to be highly reproducible with low intra-and inter-assay variation. Coefficients of variation (CV) values were 0.70-1.39% and 0.98-2.1%, respectively. Importantly, the method was applied to simultaneously quantify both plus-stranded and minus-stranded FMDV RNA using tagged-RT and tagged-FP primer during a high-temperature reverse transcription. Highly sensitive and strand-specific real-time RT-PCR assay has been established. We tested the ratio of viral plus-stranded to minus-stranded RNA in acutely infected and persistently infected BHK-21cells, for which the values ranged from 22/1 to 143/1 and from 287/1 to 334/1, respectively, suggesting different replication patterns of plus-and minus-stranded RNA in acutely infected and persistently infected cells. This value ranged from 83/1 to 93/1 in enriched FMDV virions, indicating that FMDV encapsidation is highly specific for plus-stranded RNAs. In addition, the method was applied to surveille the FMDV replication at animal level.

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