Plus- and minus-stranded foot-and-mouth disease virus RNA quantified simultaneously using a novel real-time RT-PCR.
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-21 cells, 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.
Gu
Chaojiang
C
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Zheng
Congyi
C
Shi
Lili
L
Zhang
Qian
Q
Li
Yong
Y
Lu
Bin
B
Xiong
Yi
Y
Qu
Sanpu
S
Shao
Junjun
J
Chang
Huiyun
H
D000818:Animals; D000833:Animals, Suckling; D002478:Cells, Cultured; D003199:Computer Systems; D006224:Cricetinae; D005536:Foot-and-Mouth Disease / Q000235:genetics / Q000473:pathology / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D051379:Mice; D008954:Models, Biological; D015345:Oligonucleotide Probes / Q000737:chemistry; D016372:RNA, Antisense / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D016372:RNA, Antisense; D012367:RNA, Viral