Cycling probe‐based real‐time PCR for the detection of Human herpesvirus 6A and B. Issue 9 (16th March 2016) - Ihira, Masaru
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.