Optimisation of reverse transcription can improve the sensitivity of RT-PCR for detection of classical swine fever virus.
Classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the World Organisation for Animal Health (OIE). Therefore, methods employed in the diagnosis of CSF should be fast, sensitive and specific. The aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time RT-PCR for the detection of classical swine fever virus, the aetiological agent of the disease. The efficiency of reverse transcription reaction was compared including a range of reverse transcriptases, thermal conditions and priming methods based on results obtained in the following realtime PCR. Depending on catalysis and the priming method used in the study a significant diversity of results was observed. The best efficacy of reverse transcription was obtained using SuperScript II reverse transcriptase and priming with random nonamers and reverse, gene-specific primer. This combination improved the sensitivity of RT-PCR nearly 1000 times as compared to the method with AMV reverse transcriptase coupled with random hexamers. In summary, this study has demonstrated that the optimisation of reverse transcription can contribute to a higher sensitivity of RT-PCR diagnostic methods.
Podgórska
Katarzyna
K
National Veterinary Research Institute, Swine Diseases Department, Al. Partyzantów 57, 24-100 Puławy, Poland. katarzyna.podgorska@piwet.pulawy.pl
Stadejek
Tomasz
T
D006692:Classical Swine Fever Virus / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013696:Temperature
D012194:RNA-Directed DNA Polymerase