儿童急性呼吸道感染中腺病毒环介导等温扩增技术检测方法的建立

Detecting human adenoviruses in respiratory samples collected from children with acute respiratory infections by loop-mediated isothermal amplification

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1．9×10^2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宴聆窜懊谏榆测．具右糖好的章际席用前罱．

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 Adl4）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 cross- reaction 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 x 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.