Detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral L1 genome segment.
A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.
Leary
Thomas P
TP
Virus Discovery Group, Abbott Diagnostics Division, Abbott Laboratories, Experimental Biology Research, Department 90D, Building L3, 1401 Sheridan Road, North Chicago, IL 60064-6269, USA.
Erker
James C
JC
Chalmers
Michelle L
ML
Wetzel
J Denise
JD
Desai
Suresh M
SM
Mushahwar
Isa K
IK
Dermody
Terence S
TS
D002021:Buffers; D016679:Genome, Viral; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012087:Reoviridae / Q000235:genetics / Q000302:isolation & purification*; D012088:Reoviridae Infections / Q000175:diagnosis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D002021:Buffers; D012367:RNA, Viral