Preliminary report on a single-tube, non-interrupted reverse transcription-polymerase chain reaction for the detection of rabies virus in brain tissue.
A simple method for the rapid detection of rabies virus was developed employing a single-tube reverse transcription polymerase chain reaction (RT-PCR). The method utilized a single buffer system for both RT and PCR and was performed without interruption as a single thermal cycling programme. Two primer sets within the genes coding for rabies nucleoprotein and glycoprotein were used to amplify a 533 bp and a 406 bp amplicon, respectively. The amplified products were detected with a challenge virus strain (CVS) of rabies. There was no amplified product with uninfected mouse or dog brain. The method was applied to detect rabies virus in 10 mouse inoculation test (MIT)-positive and three MIT-negative brain tissue samples. The amplified product was found only in the MIT-positive samples. The amplified product was confirmed by restriction endonuclease analysis using Hinf1. The results from RT-PCR correlated well with the results from MIT. This indicates that the single-tube RT-PCR may be a useful method for detecting rabies virus in brain tissue samples from suspected cases of rabies.
Gupta
P K
PK
National Biotechnology Centre, Izatnagar, India. praveen@ivri.up.nic.in
Singh
R K
RK
Sharma
R N
RN
Rao
Y U
YU
Butchaiah
G
G
D000818:Animals; D001921:Brain / Q000821:virology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004283:Dog Diseases / Q000175:diagnosis* / Q000821:virology; D004285:Dogs; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D051379:Mice; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D011818:Rabies / Q000175:diagnosis / Q000821:virology*; D011820:Rabies virus / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*
D012367:RNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases