逆转录-环介导等温扩增技术检测急性呼吸道感染儿童中的呼吸道合胞病毒

Detecting human respiratory syncytial virus in respiratory samples collected from children with acute respiratory infections by reverse transcription-loop mediated isothermal amplification

目的利用逆转录一环介导等温扩增技术（reverseloop—mediatedisothermalamplification，RT—LAMP），建立一种快速且敏感、特异的从急性呼吸道感染儿童的呼吸道标本中检测呼吸道合胞病毒（RSV）的方法。方法设计特异性RT—LAMP扩增引物，对引物进行特异性与灵敏度评估后，应用LA-320C实时浊度仪，对RSV5株分离株及经直接免疫荧光法（DFA）呼吸道病毒多病原检测过的急性呼吸道感染患儿的呼吸道标本101例进行RT—LAMP检测，并以本研究室已建立的RT-巢式PCR为对照方法。结果所建立的RT—LAMP方法敏感度好，60min扩增反应时间内每个反应可检测到1个拷贝的RSV（A或B亚型）RNA；且方法特异性好，与其他病毒间无交叉反应；4株A亚型及1株B亚型RSV分离株RT-LAMP方法检测均为阳性；在101例经DFA检测的呼吸道标本中，所建立的RT—LAMP方法与DFA方法在RSVA、B亚型检测的总符合率为95．0％，且两种方法的相关系数为0．895；与RT-巢式PCR的总符合率为94．1％，两种方法的相关系数为0．871。结论本研究所建立的RT—LAMP方法耗时短、操作简便、灵敏且特异，适用于儿科临床工作中疑似RSV感染的呼吸道标本的快速、特异的病原学检测。

Objective 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. Method 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. Result Sensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/p^l 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. Conclusion A new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, whichshould be helpful in rapid detection of RSV from respiratory tract samples of children.