Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR
Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR
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DOI：
10.1128/jcm.41.1.149-154.2003
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发表时间：
2003-01-01
影响因子：
9.4
通讯作者：
Cheng, SM
中科院分区：
文献类型：
--
作者：
Hu, AZ;Colella, M;Cheng, SM
呼吸道合胞病毒（RSV）感染的及时诊断对于幼儿下呼吸道感染的适当治疗至关重要。为了便于诊断，我们开发了一种快速、特异、敏感的TaqMan PCR方法用于检测RSV A和RSV B。从编码核衣壳蛋白的核苷酸序列中选择两组引物-探针对-一组靶向RSV A，另一组靶向RSV B。通过检测各引物-探针对来自实验室病毒储备液以及临床呼吸道标本的各种病毒的特异性，评价TaqMan逆转录-PCR检测试剂盒的特异性。仅在存在RSV A和/或RSV B的情况下观察到荧光信号。根据PFU和病毒颗粒计数确定定量PCR检测的灵敏度。所得试验灵敏度为0.023 PFU（RSV A）或2个病毒RNA拷贝，0.018 PFU（RSV B）或9个病毒RNA拷贝。应用TaqMan定量PCR方法对2000年和2001年冬季香港175例有呼吸道症状儿童的鼻咽吸出物进行了诊断。在这些标本中，TaqMan PCR检测到36份RSV阳性标本，其中10份鉴定为RSV A，26份鉴定为RSV B，而培养确认鉴定出21份RSV阳性标本，免疫荧光鉴定出32份RSV阳性标本，所有这些标本均在PCR鉴定的标本中。结果证实了我们的TaqMan PCR检测的准确性，并证明了其相对于经典方法的灵敏度提高。
Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan PCR method for detection of RSV A and RSV B. Two sets of primer-probe pairs were selected from the nucleotide sequences encoding the nucleocapsid protein-one targeting RSV A and the other targeting RSV B. The specificity of the TaqMan reverse transcription-PCR assay was evaluated by testing each primer-probe pair against various viruses derived from laboratory virus stocks, as well as clinical respiratory specimens. Fluorescent signals were observed only in the presence of RSV A and/or RSV B. The sensitivity of our quantitative PCR assay was determined on the basis of PFU and virus particle counts. The resulting assay sensitivity was found to be 0.023 PFU, or two copies of viral RNA, for RSV A and 0.018 PFU, or nine copies of viral RNA, for RSV B. This quantitative TaqMan PCR assay was utilized to diagnose 175 nasopharyngeal aspirates obtained from children in Hong Kong with respiratory symptoms during the winter of 2000 and 2001. Among these specimens, TaqMan PCR detected 36 RSV-positive samples, 10 of which were identified as RSV A and 26 of which were identified as RSV B, whereas culture confirmation identified 21 RSV-positive specimens and immunofluorescence identified 32 RSV-positive specimens, all of which were among those identified by PCR. The results confirmed the accuracy of our TaqMan PCR assay and demonstrated its improved sensitivity versus classical methods.