title: Cycling probe-based real-time PCR for the detection of Human herpesvirus 6A and B.
abstract: Human herpesvirus 6 (HHV-6) is classified as two distinct species: HHV-6A and B. HHV-6B infection can cause several clinical manifestations in transplant recipients including encephalitis, bone marrow suppression, and pneumonitis. In contrast to HHV-6B, the clinical features of HHV-6A infection remain largely undefined. Herein, we developed a multiplex cycling probe real-time PCR that discriminated between HHV-6A and HHV-6B. The assay was HHV-6-specific and no cross amplification was demonstrated for other herpesviruses. Moreover, the assay had a broad, linear dynamic range of detection between 1 and 10(6) copies of viral DNA. The quantification of HHV-6A DNA was suppressed by an excess amount of HHV-6B DNA (1 × 10(6) copies/tube) in the multiplex PCR assay; however, 1 × 10(6) copies/tube of HHV-6A DNA did not affect the quantification of 1 × 10(4) copies/tube of HHV-6B DNA. To determine the reliability of the assay for analysis of clinical specimens, DNAs extracted from the peripheral blood of hematopoietic stem cell transplant recipients were assayed using our multiplex real-time PCR versus the standard TaqMan PCR. Strong correlations were demonstrated between the two different assay systems for both HHV-6A (R(2)  = 0.913) and HHV-6B (R(2)  = 0.909). Therefore, our multiplex HHV-6 species-specific cycling probe real-time PCR is useful for evaluating the precise copy numbers of HHV-6A and B in transplant recipients. However, as HHV-6A copy numbers was affected by presence of high copies of HHV-6B DNA (1 × 10(6) copies/tube), it may be difficult to measure precise copy numbers of HHV-6A in inherited chromosomally integrated HHV-6B patient. J. Med. Virol. 88:1628-1635, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Faculty of Clinical Engineering, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Ihira
authors.affiliation: ME Center, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Yamaki
authors.affiliation: Department of Clinical Laboratory, Fujita Health University Hospital, Toyoake, Aichi, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Clinical Laboratory, Fujita Health University Hospital, Toyoake, Aichi, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Higashimoto
authors.affiliation: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Kawamura
authors.affiliation: Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Yoshikawa
mesh_terms: D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D019349:Roseolovirus Infections / Q000821:virology; D013045:Species Specificity; D019562:Viral Load
chemical_list: D004279:DNA, Viral
keywords: HHV-6; HSCT; cycling probe; real-time PCR; variant specific