Li Fan, Zhao Lin-Qing, Qian Yuan, Deng Jie, Zhu Ru-Nan, Sun Yu, Liu Li-Ying
Laboratory of Virology, Capital Institute of Pediatrics, Beijing 100020, China.
Zhonghua Er Ke Za Zhi. 2013 Apr;51(4):270-5.
To establish a rapid, sensitive and specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detecting human respiratory syncytial virus (RSV) in respiratory samples collected from children with acute respiratory infections.
According to the conserved matrix gene sequences of respiratory syncytial virus subtypes A and B downloaded from GenBank, primers were designed and RT-LAMP assay was developed to detect RNA of RSV sensitivity of the RT-LAMP method was evaluated by using ten-fold serially diluted in vitro-transcribed matrix RNA fragments from RSV A and RSV B, respectively. Specificity of the RT-LAMP method was tested through cross-reaction with other RNA and DNA viruses. Then 5 RSV strains isolated from clinical specimens using tissue cultures were tested by RT-LAMP assay. A total of 101 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 40 positive for RSV and 61 negative for RSV, were tested by RT-LAMP assay and by RT-nested PCR.
Sensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/µl of RSV A and RSV B RNA, no amplification was shown in RT-LAMP with DNA or cDNA from other viruses in 60 min, revealed that the RT-LAMP assay is highly specific. Five RSV isolates confirmed as 4 RSV A and 1 RSV B previously were detected by RT-LAMP method as positive in 30 min. For those 101 specimens tested, 37 were RSV positive determined by RT-LAMP assay, as well as 35 RSV positive by RT-nested PCR. The total coincidence rate of RT-LAMP assay with DFA and RT-nested PCR in detecting RSV is 95.0%, 94.1% with Kappa value 0.895 and 0.871, respectively.
A new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, which should be helpful in rapid detection of RSV from respiratory tract samples of children.
建立一种快速、灵敏且特异的逆转录环介导等温扩增（RT-LAMP）检测方法，用于检测从急性呼吸道感染儿童采集的呼吸道样本中的人呼吸道合胞病毒（RSV）。
根据从GenBank下载的呼吸道合胞病毒A、B亚型保守基质基因序列设计引物，建立RT-LAMP检测方法以检测RSV的RNA。分别使用从RSV A和RSV B体外转录的基质RNA片段进行十倍系列稀释，评估RT-LAMP方法的灵敏度。通过与其他RNA和DNA病毒的交叉反应测试RT-LAMP方法的特异性。然后使用RT-LAMP检测方法对通过组织培养从临床标本中分离出的5株RSV菌株进行检测。对101例已通过直接免疫荧光法（DFA）检测的住院急性呼吸道感染患者的鼻咽抽吸物进行检测，其中包括40例RSV阳性和61例RSV阴性，分别通过RT-LAMP检测方法和RT巢式PCR进行检测。
灵敏度分析表明，该RT-LAMP方法能够检测到1拷贝/μl的RSV A和RSV B RNA；在60分钟内，使用其他病毒的DNA或cDNA进行RT-LAMP检测未出现扩增现象，表明RT-LAMP检测方法具有高度特异性。先前确认的5株RSV分离株，其中4株为RSV A，1株为RSV B，通过RT-LAMP检测方法在30分钟内检测为阳性。对于所检测的101份标本，RT-LAMP检测方法确定37份为RSV阳性，RT巢式PCR确定35份为RSV阳性。RT-LAMP检测方法与DFA和RT巢式PCR在检测RSV方面的总符合率分别为95.0%、94.1%，Kappa值分别为0.895和0.871。
建立了一种用于从鼻咽抽吸物中检测人呼吸道合胞病毒的新的、灵敏、准确且快速的方法——RT-LAMP检测方法，这将有助于从儿童呼吸道样本中快速检测RSV。
