Development of in situ nest PCR and comparison of five molecular biological diagnostic methods for the detection of intracellular viral DNAs in paraffin sections.
Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10(1) to 10(5) 50% tissue culture infected doses (TCID(50)), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions.
Kim
Okjin
O
USDA-ARS ADRU, Pullman, WA 99164-7030, USA.
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D004847:Epithelial Cells / Q000821:virology; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000166:cytology / Q000821:virology; D008867:Microtomy; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral