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Development of real‐time RT‐PCR assays for detection of three classes of HHV‐6A gene transcripts
Author(s) -
Ihira Masaru,
Urashima Akiko,
Miura Hiroki,
Hattori Fumihiko,
Kawamura Yoshiki,
Sugata Ken,
Yoshikawa Tetsushi
Publication year - 2017
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.24862
Subject(s) - real time polymerase chain reaction , biology , virology , viremia , peripheral blood mononuclear cell , gene , polymerase chain reaction , reverse transcription polymerase chain reaction , microbiology and biotechnology , plasmid , reverse transcriptase , virus , gene expression , genetics , in vitro
Human herpesvirus 6 (HHV‐6), a member of the betaherpesvirus family, has two distinct species: HHV‐6A and HHV‐6B. HHV‐6B real‐time reverse transcription polymerase chain reaction (RT‐PCR) has been used to distinguish between active and latent viral infection. In this study, we developed a real‐time RT‐PCR assay to detect HHV‐6A‐specific transcripts and evaluated its reliability for analysis of clinical samples. To develop HHV‐6A‐specific real‐time RT‐PCR assays, three different classes of gene transcripts (immediate early: U90; early: U12; and late: U100) were selected as targets. Serial d ilutions of plasmid DNAs containing target sequences and RNAs extracted from HHV‐6A‐infected cells were used to determine assay specificity and sensitivity. Peripheral blood mononuclear cells (PBMCs) collected from patients with either primary or reactivated HHV‐6B infection, and one patient with X‐linked severe combined immunodeficiency (X‐SCID) with HHV‐6A reactivation, were used to evaluate assay reliability. The HHV‐6A‐specific real‐time RT‐PCR assays amplified plasmids containing the target sequences at concentrations between 10 and 1 × 10 6 copies per reaction. The intra‐assay coefficients of variation were less than 5%. The three classes of HHV‐6A gene transcripts were not detected in any HHV‐6B sample isolated from the patients. In the X‐SCID patient, high copy numbers of HHV‐6A U12 and U100 transcripts were detected in PBMC samples during viremia. Thus, we successfully established highly sensitive and reproducible real‐time RT‐PCR methods targeting three classes of HHV‐6A gene transcripts. This method should be useful for discriminating active HHV‐6A infection from either latent infection or chromosomally integrated HHV‐6A (ciHHV‐6A).