title: Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts.
abstract: The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100 ng/reaction) and C(t) value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection.
authors: Faculty of Clinical Engineering, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
authors: Masaru
authors: M
authors: Ihira
authors: Yoshihiko
authors: Y
authors: Enomoto
authors: Yoshiki
authors: Y
authors: Kawamura
authors: Hidetaka
authors: H
authors: Nakai
authors: Ken
authors: K
authors: Sugata
authors: Yoshizo
authors: Y
authors: Asano
authors: Motohiro
authors: M
authors: Tsuzuki
authors: Nobuhiko
authors: N
authors: Emi
authors: Tatsunori
authors: T
authors: Goto
authors: Koichi
authors: K
authors: Miyamura
authors: Kimikazu
authors: K
authors: Matsumoto
authors: Koji
authors: K
authors: Kato
authors: Yoshiyuki
authors: Y
authors: Takahashi
authors: Seiji
authors: S
authors: Kojima
authors: Tetsushi
authors: T
authors: Yoshikawa
mesh_terms: D000328:Adult; D002648:Child; D002675:Child, Preschool; D005077:Exanthema Subitum / Q000175:diagnosis* / Q000821:virology; D005260:Female; D005814:Genes, Viral; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014775:Virus Activation; D055815:Young Adult
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral