实时荧光重组酶聚合酶扩增技术(RPA)快速检测鼠疫耶尔森菌  

A real-time recombinase polymerase amplification(RPA)assay for rapid detection of Yersinia pestis

作　　者：白欣茹 袁媛 徐健皓 辛文文 康琳 王菁 李岩伟 高姗 赵宝华[1] 王景林 BAI Xin-ru;YUAN Yuan;XU Jian-hao;XIN Wen-wen;KANG Lin;WANG Jing;LI Yan-wei;GAO Shan;ZHAO Bao-hua;WANG Jing-lin(College of Life Sciences,Hebei Normal University,Shijiazhuang 050024,China;State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou,350000,China)

机构地区：[1]河北师范大学生命科学学院,石家庄050024 [2]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [3]福建农林大学生命科学学院,福州350000

出　　处：《军事医学》2022年第7期487-492,共6页Military Medical Sciences

摘　　要：目的建立基于实时荧光重组酶聚合酶扩增(RPA)的鼠疫耶尔森菌快速检测技术。方法以鼠疫菌特异基因序列YPS723为靶标,设计实时荧光RPA引物探针并进行筛选;构建含有基因待检片段的重组质粒进行实时荧光RPA方法灵敏度的评价;将15种非鼠疫菌细菌基因组混合后进行实时荧光RPA方法特异性验证;将鼠疫菌基因组掺入人血清和小鼠全血中模拟自然感染状态,以评估实时荧光RPA检测的实用性。结果筛选出的实时荧光RPA引物、探针在39℃条件下15 min内即可在便携装置上获得检测结果,最低检测模板质粒浓度为10拷贝/μl,与其他细菌无交叉反应且重复性良好。实时荧光RPA对人血清模拟样本和小鼠全血模拟样本中鼠疫菌基因组的检出限为50 fg/μl,且在模拟样本盲测中正确率达100%。结论该研究开发了一种快速、便携、灵敏度高、特异性好的实时荧光RPA方法用于鼠疫菌的检测。Objective To establish a rapid technology for the detection of Yersinia pestis based on real-time recombinase polymerase amplification(RPA).Methods The real-time RPA primers and probes were designed using YPS723 gene of Yersinia pestis.A recombinant plasmid containing targeted regions was constructed to assess the sensitivity of the real-time RPA method.The analytical specificity was tested using the DNA mixture of 15 kinds of non-Yersinia pestis bacteriaDNA extracts.Moreover,Yersinia pestis DNA was spiked into human serum and mouse blood to mimic natural infection samples and to evaluate the feasibility of real-time RPA detection.Results Real-time RPA assay could be finished on a portable device within 15 minutes at 39℃.The minimum detection concentration of plasmid was 10 copies/μl.There was no cross-reaction with other bacteria and the repeatability was good.The detection sensitivity of real-time RPA for Yersinia pestis genome in human serum and mouse blood samples was 50 fg/μl,and the detection accuracy was as high as 100%in the detection of simulated samples.Conclusion This research has developed a fast,portable,sensitive and specific real-time RPA method to detect Yersinia pestis.

关 键 词：鼠疫耶尔森菌 实时荧光RPA YPS723 检测 

分 类 号：Q93-331[生物学—微生物学]