Development of real-time RT-PCR assays for detection of three classes of HHV-6A gene transcripts
Development of real-time RT-PCR assays for detection of three classes of HHV-6A gene transcripts
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开发用于检测三类 HHV-6A 基因转录本的实时 RT-PCR 检测方法
DOI：
10.1002/jmv.24862
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发表时间：
2017
影响因子：
12.7
通讯作者：
Yoshikawa T
中科院分区：
文献类型：
--
作者：
Ihira M;Urashima A;Miura H;Hattori F;Kawamura Y;Sugata K;Yoshikawa T
人类疱疹病毒6型(HHV-6)是β疱疹病毒家族的成员，有两个不同的物种：HHV-6A和HHV-6B。HHV-6B实时逆转录聚合酶链式反应(RT-PCR)已被用于区分病毒感染的活动性和潜伏性。在这项研究中，我们建立了一种实时RT-PCR方法来检测HHV-6A特异性转录本，并评估了其用于临床样本分析的可靠性。为了建立针对HHV-6A的实时荧光RT-PCR检测方法，选择了三种不同类型的基因转录本(即刻早期：U90；早期：U12；晚期：U100)作为靶标。连续稀释含有靶序列的质粒DNA和从HHV-6A感染细胞中提取的RNA，以确定检测的特异性和敏感性。用采集自HHV-6B初次感染和再激活患者以及1例X连锁严重联合免疫缺陷(X-SCID)患者和HHV-6A重新激活的患者的外周血单个核细胞(PBMC)来评价检测的可靠性。HHV-6A特异性实时RT-PCR法在每次反应的浓度为10到1 × 106拷贝之间时扩增出含有目的序列的质粒。批内变异系数小于5%。从患者分离的HHV-6B样本中均未检测到这三类HHV-6A基因转录本。在X-SCID患者中，病毒血症患者的PBMC中检测到高拷贝数的HHV-6A U12和U100转录本。因此，我们成功地建立了针对三类HHV-6A基因转录本的高灵敏度和可重复性的实时RT-PCR方法。该方法可用于区分活动性HHV-6A感染和潜伏感染或染色体整合HHV-6A(CIHHV-6A)。
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 × 106copies 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).