Detection and quantitation of group A rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction.
A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to detect and to quantitate the RNA of group A rotaviruses. In the assay, a 433 bp fragment is amplified by a one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment 6 of the rotavirus genome. An in vitro synthesized RNA with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. Using these transcripts as templates, 10 RNA molecules were amplified reproducibly and detected in ethidium bromide-stained agarose gels or by fluorimetry using the SYBR Green I dye in a real-time RT-PCR assay. The efficiency of the protocol was confirmed by the detection of small amounts of viral RNA of group A rotaviruses in clinical samples obtained from various animal species and man.
Schwarz
Bernd-Andreas
BA
Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, 04103, Leipzig, Germany.
Bange
Reinhard
R
Vahlenkamp
Thomas W
TW
Johne
Reimar
R
Müller
Hermann
H
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
D017931:DNA Primers; D012367:RNA, Viral