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。
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.
作者
王秋平
黄恩奇
杨银梅
钟志敏
陈斌
WANG Qiu-ping;HUANG En-qi;YANG Yin-mei;ZHONG Zhi-min;CHEN Bin(The First Affiliated Hospital of University of South China,Hengyang,Hunan 421001,China)
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2018年第24期3734-3737,共4页
Chinese Journal of Nosocomiology
基金
国家自然科学基金资助项目(81201163)
广东省医学科研基金资助项目(A2018122)
广州市科学研究计划基金资助项目(201804010333)
关键词
重组酶聚合酶扩增技术
烟曲霉菌
DNA提取
快速检测
Recombinase polymerase amplification
Aspergillus fumigatus
DNA extraction
Rapid detection
