Development of in situ Nest PCR and Comparison of Five Molecular Biological Diagnostic Methods for the Detection of Intracellular Viral DNAs in Paraffin Sections.

Abstract

ABSTRACT. 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. KEY WORDS: in situ hybridization, in situ PCR, in situ PCR/hybridization, in situ nest PCR.

A highly sensitive molecular diagnosis method for detecting Toxoplasma gondii tachyzoite: a PCR/dot blot hybridization

“…For DBH analysis, T. gondii-specific DNA probes were prepared by digoxigenin (DIG)-labeling after amplification of the genomic DNA by PCR as described previously (Kim, 2003). To prepare T. gondii-specific DNA probes, the PCR products amplified with ITS-1 primers were purified using Wizard PCR preps (Promega, Medison, WI, USA) and then labeled by random priming with DIG-dUTP (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.…”