Detection of viral infections by in situ PCR: theoretical considerations and possible value in diagnostic pathology.
The technique that allows for in situ detection of PCR-amplified DNA and cDNA has improved remarkably quickly over the past 5 years. As of this writing, one can expect good results after a relatively quick "learning curve." The technique demands some skill in working with glass slides and cells/tissues. Thus, I recommend that one without experience in tissue analysis spend a few weeks working with either standard in situ hybridization or immunohistochemistry to be comfortable in that regard. Commercially available kits for in situ hybridization are good and readily available (e.g., Enzo Diagnostics, NY). When ready to begin in situ PCR, the most user-friendly system now available is from Perkin Elmer. I cannot overstate the importance of performing the needed controls with each and every run, of the crucial relationship between protease digestion time and success with RT in situ PCR, and the need to use relatively high (4.5 mM) MgCl2 and to include bovine serum albumin (or increased Taq polymerase). Given these improvements and the important information available from in situ PCR that cannot be provided by standard in situ hybridization or solution-phase PCR, one would anticipate that in situ PCR will someday enter the field of molecular diagnostics. This need not be restricted to the field of infectious diseases. A recent study using RT in situ PCR showed that determination of the metalloprotease to tissue inhibitor of metalloprotease expression in cancers was strongly correlated with the clinical behavior of the tumor (30).
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GJ
MGN Medical Research Laboratory, Setauket, New York 11733, USA.
D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D006651:Histocytochemistry; D006801:Humans; D017403:In Situ Hybridization; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D014407:Tumor Cells, Cultured; D014777:Virus Diseases / Q000175:diagnosis*
D018076:DNA, Complementary; D004279:DNA, Viral; D010450:Endopeptidases