Differentiation between porcine epidemic diarrhea virus and transmissible gastroenteritis virus in formalin-fixed paraffin-embedded tissues by multiplex RT-nested PCR and comparison with in situ hybridization.
Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were detected and differentiated in formalin-fixed, paraffin-embedded tissues from experimentally and naturally infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). The results of this new method were compared with in situ hybridization. A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of a suitable quality for reliable and consistent multiplex RT-nPCR analyses. PEDV and TGEV cDNAs were detected in jejunal tissues from experimentally and naturally infected pigs by multiplex RT-nPCR. Distinct positive signals for PEDV and TGEV were also detected in the same jejunal tissues by in situ hybridization. The rate of conformity between multiplex RT-nPCR and in situ hybridization was 100% for the detection of PEDV and TGEV in formalin-fixed paraffin-embedded jejunal tissues.
Jung
Kwonil
K
Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, Kwanak-Gu 151-742, South Korea.
Kim
Junghyum
J
Kim
Okjin
O
Kim
Bongtae
B
Chae
Chanhee
C
D000818:Animals; D001483:Base Sequence; D017934:Coronavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D000086862:Coronavirus M Proteins; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005557:Formaldehyde; D005761:Gastroenteritis, Transmissible, of Swine / Q000453:epidemiology / Q000821:virology; D017403:In Situ Hybridization; D007583:Jejunum / Q000821:virology; D017720:Molecular Epidemiology; D016612:Paraffin Embedding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D034421:Sus scrofa; D016707:Tissue Fixation; D005760:Transmissible gastroenteritis virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D014763:Viral Matrix Proteins / Q000235:genetics; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D000086862:Coronavirus M Proteins; D017931:DNA Primers; D004279:DNA, Viral; C000706236:M protein, Porcine epidemic diarrhea virus; C000706238:M protein, Transmissible gastroenteritis virus; D014763:Viral Matrix Proteins; D005557:Formaldehyde