Development of an optimized protocol for the detection of classical swine fever virus in formalin-fixed, paraffin-embedded tissues by seminested reverse transcription-polymerase chain reaction and comparison with in situ hybridization.
An optimized protocol was developed for the detection of classical swine fever virus (CSFV) in formalin-fixed, paraffin-embedded tissues obtained from experimentally and naturally infected pigs by seminested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that the use of deparaffinization with xylene, digestion with proteinase K, extraction with Trizol LS, followed by seminested RT-PCR is a reliable detection method. An increase in sensitivity was observed as amplicon size decreased. The highest sensitivity for RT-PCR on formalin-fixed, paraffin-embedded tissues RNA was obtained with amplicon sizes less than approximately 200 base pairs. An hybridization signal for CSFV was detected in lymph nodes from 12 experimentally and 12 naturally infected pigs. The sensitivity of seminested RT-PCR compared with in situ hybridization was 100% for CSFV. When only formalin-fixed tissues are available, seminested RT-PCR and in situ hybridization would be useful diagnostic methods for the detection of CSFV nucleic acid.
Ha
S-K
SK
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu 151-742, Seoul, Republic of Korea.
Choi
C
C
Chae
C
C
D000818:Animals; D006691:Classical Swine Fever / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D019286:Endopeptidase K; D005404:Fixatives; D005557:Formaldehyde; D006146:Guanidines; D017403:In Situ Hybridization / Q000662:veterinary*; D008198:Lymph Nodes / Q000821:virology; D016612:Paraffin Embedding / Q000662:veterinary; D010636:Phenols; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D013552:Swine; D016707:Tissue Fixation / Q000662:veterinary; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D005404:Fixatives; D006146:Guanidines; D010636:Phenols; D012367:RNA, Viral; C411644:trizol; D005557:Formaldehyde; D019286:Endopeptidase K