You Huey-Ling, Chang Shun-Jen, Yu Hong-Ren, Li Chia-Chin, Chen Chang-Han, Liao Wei-Ting
Departments of Laboratory Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan.
BMC Pediatr. 2017 Mar 27;17(1):89. doi: 10.1186/s12887-017-0843-7.
Both respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance.
In order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay.
Our one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 10-10copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously.
In summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.
呼吸道合胞病毒（RSV）和人偏肺病毒（hMPV）都是引起儿科人群呼吸道感染（RTI）的重要病毒病原体。然而，RSV和hMPV感染的临床表现相似。因此，需要一种可靠且快速的诊断工具来提高诊断效能。
为了优化RTI的诊断效率，本研究的目的是建立一种快速且先进的方法，用于同时检测患者鼻咽抽吸物标本中的RSV和hMPV。我们设计了一种使用TaqMan探针的一步三重实时逆转录聚合酶链反应（qRT-PCR）方案来检测RSV和hMPV。构建了包含RSV核蛋白基因和hMPV融合基因的质粒克隆作为参考标准。我们使用来自86例已知儿科RTI患者的病毒培养上清液来测试我们检测方法的特异性和敏感性。然后我们使用了总共222份来自有呼吸道症状住院儿科患者的鼻咽抽吸物标本对我们的检测方法进行评估。
我们的一步三重qRT-PCR检测方法在检测86份已知病毒培养上清液中的RSV和hMPV时显示出100%的敏感性和特异性，具有出色的线性（R＞0.99）和可靠的重复性（变异系数低于1.04%）。该检测方法具有10-10拷贝/反应的宽动态范围（检测限；LOD = 100拷贝/反应）。总共222例有呼吸道症状住院的患者被纳入临床评估。在这些样本中，我们的qRT-PCR检测方法检测到68例RSV阳性和18例hMPV阳性病例。然而，标准病毒培养仅检测到8例RSV阳性病例和0例hMPV病例。基于这种改进的三重qRT-PCR检测方法，我们发现RSV感染与胸部X光显示的严重炎症以及肺炎的发生有关，而这些情况以前未被观察到。
总之，我们开发了一种高度特异且敏感的一步三重qRT-PCR检测方法，可同时检测hMPV和RSV。该检测方法为常规诊断提供了一个有价值的工具。
