Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children.
Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children.
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DOI：
10.1016/j.jcv.2004.03.018
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发表时间：
2004-10
期刊：
影响因子：
--
通讯作者：
Morrow R
中科院分区：
文献类型：
--
作者：
Kuypers J;Wright N;Morrow R
背景：呼吸道合胞病毒（RSV）是幼儿和免疫抑制患者下呼吸道发病的主要原因。目的：为了快速准确地定量呼吸道样本中的 RSV 并确定其亚型，我们开发并评估了两种实时 RT-PCR 检测方法。研究设计：使用基质蛋白基因共有区域的引物设计定量测定，并使用聚合酶基因的引物设计RSV-A和RSV-B检测的亚型特异性测定。 2002 年 12 月至 2003 年 5 月期间提交给华盛顿大学病毒学实验室的儿科鼻洗液样本的定量 RSV RT-PCR 结果与间接荧光抗体 RSV 抗原检测测定 (FA) 的结果进行了比较。结果：RT-PCR 检测特异性较高，11 种常见呼吸道病毒和 8 种疱疹病毒均未扩增。在 751 份样本中，FA 检测出 267 份（35.6%）RSV，RT-PCR 检测出 286 份（38.1%）RSV。 FA 和 RT-PCR 均呈阳性的鼻洗液样本中 RSV 拷贝数中位数为 2.5×107 拷贝/mL，而仅通过 RT-PCR 检测呈阳性的样本中 RSV 拷贝数中位数为 3.0×104 拷贝/mL (P<0.001)。呼吸道标本中 RSV 的检测和数量与年龄相关，但与性别或住院情况无关。在西雅图队列的阳性样本中，52% 为 A 亚型，48% 为 B 亚型。在所有患者组（按年龄、性别和住院情况分层）和整个样本采集期间，检测到两种亚型的病毒载量相似。结论：这些实时 RT-PCR 检测为临床样本中 RSV 的检测、定量和分型提供了一种快速、特异且高度灵敏的替代方法。
Background: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients. Objectives: To rapidly and accurately quantify and subtype RSV in respiratory samples, we developed and evaluated two real-time RT-PCR assays. Study design: A quantitative assay was designed using primers for a consensus region of the matrix protein gene and a subtype-specific assay for RSV-A and RSV-B detection was designed using primers for the polymerase gene. Quantitative RSV RT-PCR results of pediatric nasal wash samples submitted to the University of Washington Virology Laboratory from December 2002, through May 2003, were compared to those of an indirect fluorescent antibody RSV antigen detection assay (FA). Results: Specificity of the RT-PCR assay was high, with no amplification of eleven common respiratory viruses and eight herpes viruses. Among 751 samples, RSV was detected in 267 (35.6%) by FA and in 286 (38.1%) by RT-PCR. Median RSV copy number in nasal wash samples that were positive by both FA and RT-PCR was 2.5×107 copies/mL versus a median of 3.0×104 copies/mL for samples positive by RT-PCR only (P<0.001). The detection and quantity of RSV in respiratory specimens was associated with younger age, but not with gender or hospitalization. Among positive samples from this Seattle cohort, 52% were subtype A and 48% were subtype B. Both subtypes were detected with similar viral loads among all patient groups (stratified by age, gender, and hospitalization), and throughout the specimen collection period. Conclusions: These real-time RT-PCR assays provide a rapid, specific, and highly sensitive alternative for detecting, quantifying, and subtyping RSV in clinical specimens.
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