【摘要】 目的通过建立重组酶聚合酶扩增技术（recombinase polymerase amplification,RPA）直接检测标准烟曲霉菌株的方法,探讨其无需样本前处理的可行性。方法针对标准烟曲霉核酸序列保守区域设计RPA扩增引物,使用100、10^-1、10^-2、10^-3、10^-4、10^-5麦氏浊度的标准烟曲霉菌液作为无样本前处理组进行直接RPA扩增;同时,分别采用柱式法、磁珠法、氯化苄法提取烟曲霉菌DNA作为样本前处理对照组并进行RPA扩增,最后,应用2%琼脂糖凝胶电泳对产物进行分析。结果标准烟曲霉菌的100、10^-1、10^-2及10^-3麦氏浊度均可于39℃30min内扩增出目的条带;三种提取方法结果分别为:柱氏法（13.8±0.83）ng/μl,磁珠法（11.5±2.25）ng/μl,氯化苄法（64.9±1.31）ng/μl,三种方法均能扩增出目的条带,其中氯化苄法产出率较高（64.9±1.31）ng/μl。结论 RPA可以扩增出未经前处理（核酸提取）的样本,具有快速简便的优点,为检测烟曲霉菌提供了一个有效的工具。更多还原

【Abstract】 OBJECTIVE To evaluate the feasibility of the recombinase polymerase amplification technology（RPA）in detecting Aspergillus fumigatus without sample pretreatment.METHODS RPA amplification primers were designed according to the conserved regions of A fumigatus nucleic acid sequence.The standard A fumigatus solutions at Meyer’s Turbidity of 100,10-1,10-2,10-3,10-4,10-5 were detected by RPA without sample pretreatment.The DNA templates of A fumigatus were extracted by column method,magnetic beads method and benzyl chloride method and then amplified by RPA,which were used as sample pretreatment control group.Finally,the amplified products were analyzed by 2% agarose gel electrophoresis.RESULTS The target band could be amplified from the standard A fumigatus solutions at Meyer’s Turbidity of 100,10^-1,10^-2 and 10^-3 within 30 min at 39℃.Results of the three extraction methods were:column method（13.8±0.83）ng/μl,magnetic bead method（11.5±2.25）ng/μl,and benzyl chloride method（64.9±1.31）ng/μl.All three extraction methods could amplify the target band,and the highest yield was obtained with the benzyl chloride method（64.9±1.31 ng/μl）.CONCLUSION Without sample pretreatment steps,RPA can amplify unprocessed（nucleic acid extraction）samples,with the advantages of rapid and simple process,providing an effective tool for detection of A fumigatus.更多还原