Reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections.
An assay based on reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection of hog cholera virus (HCV) and bovine virus diarrhoea virus (BVDV) in cell culture. In this study, a precipitate of the supernatants derived from cell cultures infected with HCV and BVDV was used in RT reactions, in place of extracted viral RNA. Both RT and PCR were performed using recombinant Thermus thermophilus (rTth) DNA polymerase. The specificity of the RT-PCR products was confirmed by hybridisation with a digoxygenin-labelled DNA probe. The results not only show that the stage of RNA isolation can be bypassed, but also illustrate an easy and efficient means of obtaining templates suitable for identification and characterisation of HCV and BVDV in tissue culture by RT-PCR.
Stadejek
T
T
Department of Swine Diseases, National Veterinary Research Institute, Pulawy, Poland.
Pejsak
Z
Z
Kwinkowski
M
M
Okruszek
A
A
Winiarczyk
S
S
D000818:Animals; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000821:virology; D002417:Cattle; D002460:Cell Line; D006691:Classical Swine Fever / Q000175:diagnosis* / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis / Q000737:chemistry; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D013552:Swine; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral