CAJ | 학술논문

目的利用环介导等温扩增技术(LAMP),建立一种快速可靠的儿童急性呼吸道感染标本中腺病毒检测方法.方法根据腺病毒六邻体基因序列设计特异性LAMP引物,使用实时浊度仪对所建立的LAMP方法进行特异性与灵敏度评估后,对3、7、14型腺病毒分离株共11株进行了检测,随后对经直接免疫荧光法(DFA)鉴定为腺病毒阳性的呼吸道标本36例、腺病毒阴性的呼吸道标本72例分别应用巢式多重PCR和LAMP两种方法进行检测.结果所建立的LAMP方法最低检出腺病毒DNA浓度为1.9×102拷贝/ml；方法特异性好,与其他病毒间无交叉反应；11株腺病毒分离株应用LAMP方法检测全部为阳性；36例已经过DFA鉴定为腺病毒阳性的标本中,应用LAMP法共检出腺病毒DNA阳性34例；72例DFA鉴定为腺病毒阴性的标本应用LAMP方法检测5例阳性.本研究建立的LAMP方法与DFA的总符合率为93.5％；与巢式多重PCR的总符合率为98.1％.结论本研究建立的LAMP方法耗时短、灵敏度好、特异性高、操作简便,适用于儿童呼吸道标本中腺病毒的实验室快速检测,具有较好的实际应用前景.
목적 이용배개도등온확증기술(LAMP),건립일충쾌속가고적인동급성호흡도감염표본중선병독검측방법.방법 근거선병독륙린체기인서렬설계특이성LAMP인물,사용실시탁도의대소건립적LAMP방법진행특이성여령민도평고후,대3、7、14형선병독분리주공11주진행료검측,수후대경직접면역형광법(DFA)감정위선병독양성적호흡도표본36례、선병독음성적호흡도표본72례분별응용소식다중PCR화LAMP량충방법진행검측.결과 소건립적LAMP방법최저검출선병독DNA농도위1.9×102고패/ml；방법특이성호,여기타병독간무교차반응；11주선병독분리주응용LAMP방법검측전부위양성；36례이경과DFA감정위선병독양성적표본중,응용LAMP법공검출선병독DNA양성34례；72례DFA감정위선병독음성적표본응용LAMP방법검측5례양성.본연구건립적LAMP방법여DFA적총부합솔위93.5％；여소식다중PCR적총부합솔위98.1％.결론 본연구건립적LAMP방법모시단、령민도호、특이성고、조작간편,괄용우인동호흡도표본중선병독적실험실쾌속검측,구유교호적실제응용전경.
Objective To establish a rapid and reliable loop-mediated isothermal amplification (LAMP) method for detecting adenoviruses (ADV)in respiratory samples collected from children with acute respiratory infections.Method According to the sequences of hexon genes of common adenovirus serotypes (Ad3,Ad7,and Ad14)downloaded from GenBank,primers were designed and LAMP method for detecting adenovirus DNA was developed.Sensitivity of the LAMP method was evaluated by using constructed recombinant plasmid DNA with gene fragment from hexon of ADV3,and specificity was tested through crossreaction with other viruses.Then 11 ADV strains isolated from clinical specimens using tissue cultures were tested by LAMP method.A total of 108 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA),including 36 for ADV positive and 72 for ADV negative,were tested by both LAMP method and multiplex nested PCR.Result Analysis for sensitivity indicated that this LAMP method can detect 1.9 × 102copies/ml of DNA,and no amplification was shown in DNA or cDNA of other viruses,which revealed that the specificity of the LAMP method is high.For 108 specimens which had been tested by DFA,34 out of the 36 ADV positive specimens showed positive signal within 90 minutes using LAMP.Five out of 72 negative specimens by DFA were positive using LAMP; 39 out of the 41 ADV positive specimens by multiplex nested PCR showed positive signal using LAMP,including 19 for Ad3 and 20 for Ad7; 67 negative specimens confirmed by multiplex nested PCR showed negative signal using LAMP.The total consistency rate of DFA and LAMP method for detecting ADV was 93.5％,and the total coincidence rate of multiplex nested PCR and LAMP method for detecting ADV was 98.1％.Conclusion A new,sensitive,accurate and rapid method for detecting human adenovirus from nasopharyngeal aspirates by LAMP was developed,which should be a potential method for rapid detection of ADV from respiratory tract of children in clinical diagnosis of ADV infection.