Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.
Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples.
Merkelbach
S
S
Department of Pathology, Technical University Aachen, Germany.
Gehlen
J
J
Handt
S
S
Füzesi
L
L
D003587:Cytomegalovirus / Q000235:genetics; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000473:pathology; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005404:Fixatives; D006566:Herpesviridae Infections / Q000235:genetics / Q000473:pathology; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000235:genetics / Q000473:pathology
D004279:DNA, Viral; D005404:Fixatives