铜绿假单胞菌实时荧光重组酶介导链替换核酸扩增方法建立及应用  

Development and evaluation of a real-time recombinase-aid amplification assay for rapid detection of Pseudomonas aeruginosa

作　　者：王金凤 项佳林 孙晓霞 李睿文[3] 姜彦芬 陈志敏 刘立兵 王建昌 袁万哲[3] WANG Jinfeng;XIANG Jialin;SUN Xiaoxia;LI Ruiwen;JIANG Yanfen;CHEN Zhimin;LIU Libing;WANG Jianchang;YUAN Wanzhe(Technology Center of Shijiazhuang Customs,Hebei Shijiazhuang 050051,China;Hebei Academy of Science and Technology for Inspection and Quarantine,Hebei Shijiazhuang 050051,China;College of Veterinary Medicine,Hebei Agricultural University,Hebei Baoding 071001,China)

机构地区：[1]石家庄海关技术中心,河北石家庄050051 [2]河北省检验检疫科学技术研究院,河北石家庄050051 [3]河北农业大学动物医学院,河北保定071001

出　　处：《中国食品卫生杂志》2020年第5期524-529,共6页Chinese Journal of Food Hygiene

基　　金：河北省重点研发计划项目(19226636D);河北省现代农业产业技术体系羊产业创新团队(HBCT2018140204)。

摘　　要：目的建立一种快速检测铜绿假单胞菌的实时荧光重组酶介导链替换核酸扩增(real-time RAA)方法。方法基于铜绿假单胞菌ecfX基因设计特异性引物和exo探针,通过灵敏性、特异性和疑似菌株检测评估所建立方法的有效性。结果Real-time RAA方法在39℃等温条件下反应20 min即可实现对铜绿假单胞菌的有效检测;特异性强,仅对铜绿假单胞菌出现特异性扩增;灵敏性高,对铜绿假单胞菌基因组DNA的检出限为3.0×10^3 fg/反应,对纯培养铜绿假单胞菌的检出限为1.0×10^3 CFU/反应。应用所建立的real-time RAA方法对分离的36株疑似铜绿假单胞菌进行检测,结果均为铜绿假单胞菌,同VITEK 2 Compact生化鉴定方法和real-time PCR方法结果一致。结论本试验所建立的real-time RAA方法反应快速、操作简单、可靠性高,可用于不同来源的铜绿假单胞菌的快速检测和鉴定。Objective To establish a real-time recombinase-aid amplification(RAA)method for rapid detection of Pseudomonas aeruginosa.Methods Specific primers and exo probes based on ecfX gene of P.aeruginosa were designed in this study,and the validity of the method was evaluated by sensitivity,specificity and suspected strains detection.Results Real-time RAA was performed successfully at 39℃for 20 min.Only the P.aeruginosa strains but not other bacteria were amplified,showing the good specificity.The limit of detection was 3.0×10^3 fg genomic DNA per reaction,and 1.0×10^3 CFU P.aeruginosa pure culture per reaction.The developed real-time RAA was further evaluated on 36 suspected of P.aeruginosa,which were identified successfully to be P.aeruginosa.The detection result were the same with those of a real-time PCR assay and the VITEK 2 Compact.Conclusion The developed real-time RAA assay is a rapid,simple and reliable tool for accurate detection of P.aeruginosa of diverse origins.

关 键 词：铜绿假单胞菌 ecfX基因 实时荧光重组酶介导链替换核酸扩增 等温扩增 

分 类 号：R155[医药卫生—营养与食品卫生学]