Cycling probe-based real-time PCR for the detection of human herpesvirus 6A and B.
Cycling probe-based real-time PCR for the detection of human herpesvirus 6A and B.
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基于循环探针的实时 PCR 检测人类疱疹病毒 6A 和 B。
DOI：
10.1002/jmv.24513
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Yoshikawa T.
中科院分区：
文献类型：
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作者：
Ihira M;Yamaki A;Kato Y;Higashimoto Y;Kawamura Y;Yoshikawa T.
人类疱疹病毒6 （HHV‐6）被分为两个不同的种类：HHV‐6A和HHV‐6B。HHV‐6B感染可在移植受者中引起多种临床表现，包括脑炎、骨髓抑制和肺炎。与HHV - 6B相比，HHV - 6A感染的临床特征在很大程度上仍不明确。在此，我们开发了一种多重循环探针实时PCR来区分HHV - 6A和HHV - 6B。该试验对HHV - 6特异性，对其他疱疹病毒无交叉扩增。此外，该分析具有广泛的线性动态范围，检测1至106个病毒DNA拷贝。在多重PCR试验中，过量的HHV‐6B DNA （1 × 106拷贝/管）抑制了HHV‐6A DNA的定量；而1 × 106拷贝/管的HHV‐6A DNA不影响1 × 104拷贝/管的HHV‐6B DNA的定量。为了确定该方法用于临床标本分析的可靠性，我们使用多重实时PCR与标准TaqMan PCR对从造血干细胞移植受体外周血中提取的dna进行分析。两种不同检测系统对HHV‐6A （R2= 0.913）和HHV‐6B （R2= 0.909）均有很强的相关性。因此，我们的多重HHV - 6物种特异性循环探针实时PCR可用于评估移植受体中HHV - 6A和B的精确拷贝数。然而，由于HHV - 6A拷贝数受到HHV - 6B DNA高拷贝数（1 × 106拷贝/管）的影响，在遗传性染色体整合的HHV - 6B患者中，可能难以精确测量HHV - 6A拷贝数。中华医学杂志，2016,33(4):559 - 559。©2016 Wiley期刊公司
Human herpesvirus6 (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 106copies of viral DNA. The quantification of HHV‐6A DNA was suppressed by an excess amount of HHV‐6B DNA (1 × 106copies/tube) in the multiplex PCR assay; however, 1 × 106copies/tube of HHV‐6A DNA did not affect the quantification of 1 × 104copies/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 (R2= 0.913) and HHV‐6B (R2= 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 × 106copies/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.