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

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

Content Provider	Springer Nature Link
Author	Gu, Chaojiang Zheng, Congyi Shi, Lili Zhang, Qian Li, Yong Lu, Bin Xiong, Yi Qu, Sanpu Shao, Junjun Chang, Huiyun
Copyright Year	2006
Abstract	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.
Starting Page	289
Ending Page	298
Page Count	10
File Format	PDF
ISSN	09208569
Journal	Virus Genes
Volume Number	34
Issue Number	3
e-ISSN	1572994X
Language	English
Publisher	Kluwer Academic Publishers-Plenum Publishers
Publisher Date	2006-08-22
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	FMDV Real-time RT-PCR TaqMan probe Minus-strand Plus-strand Suckling mice Virions Plant Sciences Virology Medical Microbiology
Content Type	Text
Resource Type	Article
Subject	Virology Genetics Medicine Molecular Biology

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