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.

Development of real‐time RT‐PCR assays for detection of three classes of HHV‐6A gene transcripts

“…DNA was extracted from PBMCs using QIAamp Blood Kit (Qiagen) and stored at −20°C prior to assay. HHV‐6A‐specific cycling probe‐based real‐time PCR, described previously, was used to examine the kinetics of HHV‐6A DNA copy numbers in clinical samples. Meanwhile, a previously reported real‐time PCR method was used to measure HHV‐6B DNA load.…”

Amplification chemistries in clinical virology

“…Commercial kits combining quantitative real-time PCR with CPTbased detection have been used for rapid detection and quantification of HHV-6 and can also discriminate between HHV-6A and HHV-6B [109]. CPT has also been employed for differentiating strains of VZV [110], rapid and specific detection of resistant influenza A viruses [111], and HCV [112].…”

Si̇nyal Ampli̇fi̇kasyon Tekni̇kleri̇ Ve Tanisal Vi̇roloji̇deki̇ Uygulamalari

“…Bu testte tek zincirli hedef DNA molekülü sentetik olarak üretilen ve hedef DNA ile spesifik komplementer sekans içeren bir adet kimerik DNA-RNA-DNA probu ile hibridize edilir (1) prob amplifikasyon sistemlerinde amplifikasyon ürünü birikimini saptamak üzere modifiye edilmiştir (1). Human herpesvirus 6A ve B saptanması için geliştirilen CP temelli real-time PCR testi bunun bir örneğidir (23). CP real-time PCR tekniği vahşi-tip varicella-zoster virus (VZV) ve Oka aşı kökenlerinin birbirinden ayırt edilmesi ve kantitasyonu için de kullanılmıştır (24).…”