基于重组酶聚合酶扩增和微流控的病原菌检测方法  

A Method Based on Recombinase Polymerase Amplification and Microfluidic for Pathogen Detection

作　　者：台萃 许杰[1] 欧一新 蒋海霞 孙伟 TAI Cui;XU Jie;OU Yixin;JIANG Haixia;SUN Wei(School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai,200240)

机构地区：[1]上海交通大学生命科学技术学院,上海200240

出　　处：《基因组学与应用生物学》2024年第9期1620-1631,共12页Genomics and Applied Biology

基　　金：国家自然科学基金项目(32370186);上海交通大学决策咨询课题(JCZXSJB2023-13)共同资助。

摘　　要：由病原菌感染引发的疾病严重危害人类健康,病原菌的检测技术一直备受研究者的关注。目前的病原菌检测方法存在检测周期长、检测仪器成本高、空间需求大等问题,这些问题极大地限制了资源匮乏地区的病原菌防控,因此研发一种快速准确、低成本、空间需求小的检测方法对病原菌防控具有重要意义。本研究采用磁珠法提取纯化病原菌核酸,再利用重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术扩增目标片段,最后通过荧光显色实现检测结果的可视化判定。整个实验流程被设计在一张微流控芯片上,应用于肺炎克雷伯菌(Klebsiella pneumoniae)、鲍曼不动杆菌(Acinetobacter baumannii)、铜绿假单胞菌(Pseudomonas aeruginosa)和金黄色葡萄球菌(Staphylococcus aureus)等4种重要病原菌的快速检测。结果表明,基于微流控的磁珠法经优化后可在40 min内完成DNA提取纯化,与其他常见的核酸提取方法相比,该方法所得DNA的浓度、纯度和完整性较好,可用于后续PCR等分子生物学实验;基于微流控的RPA法在39℃,20 min内可完成对病原菌特征基因片段的扩增,本研究利用该方法共检测了18株不同菌种的病原菌,与对照相比,扩增产物荧光显色差异显著,测序结果进一步验证了荧光可视化判定结果的准确性。综上,与传统检测方法相比,基于微流控的磁珠法和RPA技术结合的病原菌检测方法不仅显著缩短了检测周期,而且弥补了传统检测方法的不足。该方法具有设备要求低、空间需求小、集成性好等优势,为病原菌检测提供了一种快速、经济、简便的补充方案。Diseases caused by pathogen infections pose a serious threat to human health,and thus the technologies of pathogen detection are topics of great concern to researchers.However,current pathogen identification methods have several problems such as the long time required to obtain test reports,high detection instrument costs,and large spatial requirements,which greatly limit the prevention and control of pathogens in resource-limited settings.Thus,a fast,accurate,low-cost and small space requirement detection method is of great significance in pathogen prevention and control.In this study,the magnetic beads method was employed for DNA extraction and purification,followed by the amplification of the target sequence using the recombinase polymerase amplification(RPA)assay.Subsequently,difference in fluorescence color was employed for visual judgment.The entire experimental process was designed on a microfluidic chip and applied for the rapid detection of important pathogens such as Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus.The results showed that DNA extraction and purification were completed within 40 min using the optimized microfluidic-magnetic beads method.Compared to other nucleic acid extraction methods,the optimized method provided higher concentration,better DNA purity and integrity,which could meet the needs of subsequent molecular biology experiments such as PCR;The rapid amplifications of species-specific genes were accomplished by a microfluidic-RPA method at a constant temperature(39℃)within 20 min.The universality of the method was validated by testing of 18 clinical isolates belonging to different species in this study.There was a significant difference between the RPA products of the control and samples in fluorescence color,and the accuracy of the visual judgment results was further verified by sequencing.In conclusion,compared to traditional benchtop protocols,the microfluidic-based magnetic beads and RPA method for pathogen detection significant

关 键 词：重组酶聚合酶扩增 微流控 磁珠法 可视化 病原菌检测 

分 类 号：R446.5[医药卫生—诊断学]