摘要
建立一种同时检测沙门氏菌、大肠杆菌O157:H7和布氏杆菌的快速、灵敏、高通量的检测方法。利用特异性免疫磁珠,在37℃条件下从200 mL样液体系中循环捕获目标致病菌。磁珠液提取DNA后,对3种病原菌进行多重IMS-荧光RPA检测。结果表明:针对沙门氏菌、大肠杆菌O157:H7和布氏杆菌的检测限分别达到3.0、4.5和8.7 CFU/mL。使用新建多重IMS-荧光RPA方法对人工感染60份皮张、毛皮纺织品DNA样本进行扩增,结果显示与预期结果一致;对60份公共场所收集的皮张、毛皮、纺织品DNA样本进行扩增,结果显示有2个样本沙门氏菌阳性、1个样本大肠杆菌O157:H7阳性,3份阳性样品送测序,测序结果与GenBank检索沙门氏菌、大肠杆菌O157:H7 DNA序列一致。得出结论:建立的多重IMS-荧光RPA检测体系灵敏度、特异性、准确度符合要求,能够在2 h内完成对3种病原菌检测,可以作为快速应对此三类病原菌安全突发事件的检测手段。
This study was to establish a multiplex IMS real-time RPA method for simultaneous detection of Salmonella sp.,E coli O157 and Brucella.Target pathogens were isolated from a 200 ml sample system with specific an immunomagnetic bead based technique at 37℃.The three pathogens were tested using the multiplex IMS real-time RPA after the magnetic beads had been extracted.The results were that the limits of detection of the immunomagnetic separation(IMS)-multiplex real-time RPA(RT-RPA)method were 3.0 CFU/g for Salmonella sp.,4.5 CFU/g for E coli O157 and 8.7 CFU/g for Brucella.The new multiplex IMS real-time RPA method was used to test the textile DNA of 60 textile samples artificially infected,and the results were consistent with the expected ones.Sixty textile samples collected in public places were tested by DNA amplification;and the results showed that two samples were Salmonella positive and one was Escherichia coli O157:H7 positive.The three positive samples were then sent for sequencing with a result consistent with the DNA sequences of Salmonella and Escherichia coli O157:H7 in Gen Bank.In conclusion,the multiplex IMS real-time RPA method was established with satisfactory sensitivity,specificity and accuracy,being able to detect three pathogenic bacteria in two hours.The method may be used for rapid detection of these bacteria in emergent cases.
作者
李轲
郭华麟
禹建鹰
张淑霞
连素梅
徐超
韩国全
郭会清
LI Ke;GUO Hualin;YU Jianying;ZHANG Suxia;LIAN Sumei;XU Chao;HAN Guoquan;GUO Huiqing(Zhengzhou Customs,Zhengzhou450003,China;Sichuan Agricultural University,Ya'an625000,China;Shijiazhuang Customs,Shijiazhuang050051,China)
出处
《畜牧与兽医》
北大核心
2019年第3期106-112,共7页
Animal Husbandry & Veterinary Medicine
基金
2018年度河南省科技攻关项目(182102210226)
2017年度质检总局科技计划项目(2017IK289)
2016年度国家质检总局科技计划项目(2016IK114)
