A sensitive and specific detection of Human herpesvirus 8 by polymerase chain reaction and dot blot hybridization.
Polymerase chain reaction (PCR) is a powerful technique of detecting Human herpesvirus 8 (HHV-8), but has a limited sensitivity and specificity. A new assay of HHV-8 based on combination of PCR with dot blot hybridization (DBH) was developed and evaluated for its sensitivity and specificity. An HHV-8-specific primer pair, ORF26out was used for amplification of target DNA. When the PCR product was detected visually the limit of detection was 0.1 ng DNA isolated from HHV-8-infected BC-3 cells. For DBH, the DNA amplified with the primer pair ORF26in specific for HHV-8 was labeled with digoxigenin (DIG). This DIG-labeled probe was capable of detecting 1.0 ng of DNA isolated from HHV-8-infected BC-3 cells. On the other hand, PCR combined with DBH (PCR/DBH) was more sensitive than PCR or DBH alone and also very specific. The sensitivity of PCR/DBH was higher than that of PCR and DBH alone. The PCR/DBH assay can be applied efficiently to confirm the presence of HHV-8 in clinical samples and to differentiate specifically HHV-8 infection from other viral infections.
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USDA-ARS, ADRU, 319 Bustad, WSU, Pullman, WA 99164, USA. kimoj@netian.com
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D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral