Assessment of template quality by the incorporation of an internal control into a RT-PCR for the detection of rabies and rabies-related viruses.
A method is described to assess RNA template quality by the incorporation of a ribosomal RNA (rRNA) internal (in tube) control into a standard rabies and rabies-related virus specific RT-PCR. Specific virus and rRNA templates were co-amplified in a duplex reaction from RNA extracts derived from 60 isolates representing all six of the established lyssavirus genotypes. To ensure a wide species applicability of this technique we demonstrated that the rRNA assay was capable of functioning using the cells or tissues of 14 different mammals. Parallel studies between the duplex and the unlinked lyssavirus assay demonstrated only a minor reduction in the sensitivity of the former test. The ribosomal and viral targets (unlike beta-actin RNA) were shown to have similar degradation kinetics making rRNA amplification a good control for viral target integrity. As a consequence, the use of this system would reduce the likelihood of obtaining false negative RT-PCR results from lyssavirus infected material.
Smith
J
J
Department of Virology, Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey, UK. jemma.j.s.mith2@vla.maff.gov.uk
McElhinney
L M
LM
Heaton
P R
PR
Black
E M
EM
Lowings
J P
JP
D000818:Animals; D015139:Blotting, Southern; D001921:Brain / Q000821:virology; D002415:Cats; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D004285:Dogs; D006801:Humans; D018114:Lyssavirus / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D012335:RNA, Ribosomal / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D011818:Rabies / Q000175:diagnosis*; D011820:Rabies virus / Q000235:genetics / Q000302:isolation & purification*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D012335:RNA, Ribosomal; D012367:RNA, Viral