Sensitivity and Reproducibility of Polymerase Chain Reaction Assays for Detection of Human Herpesviruses 6 and 7

Human herpesvirus 6 (HHV-6) and 7 (HHV-7) are ubiquitous viruses that undergo latency and may become reactivated leading to cytomegalovirus reactivation, bone marrow suppression, nervous system dysfunction, graft-versus-host disease and increased mortality. The aim of this study was to identify the most sensitive and reproducible nPCR for detection of HHV-6 and HHV-7 infection and to evaluate the reproducibility of these assays in different laboratories. The sensitivity of the six previously published HHV-6 (one targeting hypothetical protein Bgp009 gene, two — large tegument protein gene, one — major binding protein gene and two targeting hypothetical Bgp071 protein gene) and four HHV-7 (targeting nuclear phosphoprotein, tegument phosphoprotein, large tegument protein and immediately early A transactivator gene) nPCRs was determined. The most sensitive HHV-6 nPCR was targeted Bgp071 protein gene, which could detect 5 genomic copies of HHV-6. The most sensitive and reproducible HHV-7 nPCR assay, targeting nuclear phosphoprotein gene, could detect 1 genomic copy of HHV-7. The reproducibility of the selected HHV-6 and HHV-7 nPCRs was evaluated in five different laboratories. The results obtained in all laboratories were identical to our results, confirming that these nPCRs are useful as assays for molecular diagnostics of HHV-6 and HHV-7 infection. Cilvēka 6. un 7. herpesvīruss (HHV-6 un HHV-7) ir plaši izplatīti vīrusi, kas pēc primārās infekcijas saglabājas latentā stāvoklī visa mūža garumā. Pie dažādiem imūnsupresīviem stāvokļiem tie var tikt reaktivēti un, savukārt, izsaukt citomegalovīrusa reaktivāciju, kaula smadzeņu supresiju, nervu sistēmas disfunkciju, "transplantāts pret saimnieku" slimību un pieaugošu mirstību. Šī darba mērķis bija atrast visjutīgākās reproducējamās polimerāzes ķēdes reakcijas (polymerase chain reaction, PCR) ar iekšējo praimēšanu HHV-6, HHV-7 infekcijas noteikšanai un apstiprināt to reproducējamību dažādās laboratorijās. Tika izvērtēta sešu iepriekš publicētu HHV-6 specifisku PCR un četru HHV-7 specifisku PCR jutība. Visjutīgākā HHV-6 noteikšanai izrādījās PCR ar praimeriem, kas komplementāri Bgp071 gēnam un kas ļāva noteikt piecas HHV-6 A un B tipa genoma kopijas. Pārējo HHV-6 specifisko PCR jutība bija zemāka - 10, 25 vai 50 HHV-6 genoma kopijas. HHV-7 specifisko PCR ar praimeriem, kas komplementāri LTP un TPP gēniem, jutība bija piecas kopijas, bet ar praimeriem, kas komplementāri IE-A transaktivatoram un NPP gēnam - viena vīr