Optimisation of reverse transcription can improve the sensitivity of RT-PCR for detection of classical swine fever virus

Katarzyna Podgórska; Tomasz Stadejek

doi:10.1556/avet.58.2010.2.11

Acta Veterinaria Hungarica

Volume/Issue:: Volume 58: Issue 2

Optimisation of reverse transcription can improve the sensitivity of RT-PCR for detection of classical swine fever virus

Authors:

Katarzyna Podgórska

Katarzyna Podgórska National Veterinary Research Institute Swine Diseases Department Al. Partyzantów 57 24-100 Puławy Poland

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Tomasz Stadejek

Tomasz Stadejek National Veterinary Research Institute Swine Diseases Department Al. Partyzantów 57 24-100 Puławy Poland

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Pages:: 257–264

Online Publication Date:: 11 May 2010

Publication Date:: 01 Jun 2010

Article Category:: Journal Article

DOI:: https://doi.org/10.1556/avet.58.2010.2.11

Keywords:: Classical swine fever virus; CSFV; optimisation; reverse transcription; RT-PCR

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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.

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Acta Veterinaria Hungarica

Print ISSN:: 0236-6290

Online ISSN:: 1588-2705

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Editorial Board

Mária BENKŐ (Acta Veterinaria Hungarica, Budapest, Hungary)
Gábor BODÓ (University of Veterinary Medicine, Budapest, Hungary)
Béla DÉNES (University of Veterinary Medicine, Budapest Hungary)
Edit ESZTERBAUER (Veterinary Medical Research Institute, Budapest, Hungary)
Hedvig FÉBEL (University of Veterinary Medicine, Budapest, Hungary)
László FODOR (University of Veterinary Medicine, Budapest, Hungary)
János GÁL (University of Veterinary Medicine, Budapest, Hungary)
Balázs HARRACH (Veterinary Medical Research Institute, Budapest, Hungary)
Peter MASSÁNYI (Slovak University of Agriculture in Nitra, Nitra, Slovak Republic)
Béla NAGY (Veterinary Medical Research Institute, Budapest, Hungary)
Tibor NÉMETH (University of Veterinary Medicine, Budapest, Hungary)
Zsuzsanna NEOGRÁDY (University of Veterinary Medicine, Budapest, Hungary)
Dušan PALIĆ (Ludwig Maximilian University, Munich, Germany)
Alessandra PELAGALLI (University of Naples Federico II, Naples, Italy)
Kurt PFISTER (Ludwig-Maximilians-University of Munich, Munich, Germany)
László SOLTI (University of Veterinary Medicine, Budapest, Hungary)
József SZABÓ (University of Veterinary Medicine, Budapest, Hungary)
Péter VAJDOVICH (University of Veterinary Medicine, Budapest, Hungary)
János VARGA (University of Veterinary Medicine, Budapest, Hungary)
Štefan VILČEK (University of Veterinary Medicine in Kosice, Kosice, Slovak Republic)
Károly VÖRÖS (University of Veterinary Medicine, Budapest, Hungary)
Herbert WEISSENBÖCK (University of Veterinary Medicine, Vienna, Austria)
Attila ZSARNOVSZKY (Szent István University, Gödöllő, Hungary)

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Acta Veterinaria Hungarica
Language	English
Size	A4
Year of Foundation	1951
Volumes per Year	1
Issues per Year	4
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ISSN	0236-6290 (Print)
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