重组酶等温扩增技术监测采供血环境中致病菌的应用研究  

Application of recombinase-isothermal amplification in monitoring bacterial pathogens in areas of blood collection and production

作　　者：朱立苇[1,2] 王拥军 丁威[1,2] 孙淼军 朱发明[1,2] 胡伟 ZHU Li-wei;WANG Yong-jun;DING Wei;SUN Miao-jun;ZHU Fa-ming;HU Wei(Zhejiang Blood Center,Hangzhou,Zhejiang 310052,China)

机构地区：[1]浙江省血液中心,浙江杭州310052 [2]浙江省血液安全研究重点实验室,浙江杭州310052

出　　处：《中国卫生检验杂志》2020年第1期43-45,共3页Chinese Journal of Health Laboratory Technology

基　　金：浙江省科技计划项目(2017C33086)

摘　　要：目的采用快速重组酶介导的等温扩增技术(RAA)监测血站采供血过程中的致病菌—金黄色葡萄球菌和溶血性链球菌。方法制备10 cfu/ml^104 cfu/ml系列浓度的2种致病菌标准菌株悬液,分别加入核酸快速裂解液中,加热提取DNA,加RAA荧光型检测试剂在小型Genchek荧光检测仪内37℃条件下识别并扩增细菌特异基因片段,20 min内实时读取荧光检测值判定结果。检测8个标准菌株验证其特异性,采用形态学-生化法和RAA法分别对8个标准菌株和86份采供血环境工艺卫生样本进行检测。结果RAA法对2种致病菌的最低检出限均为102 cfu/ml,与其他细菌无交叉反应,对采供血过程中的工艺卫生样本的检测结果与形态学-生化法相同。结论RAA技术无需分离即可快速、灵敏、特异性地检测金黄色葡萄球菌和溶血性链球菌,操作快速简便,对实验人员无特殊要求,可用于采供血环境中的致病菌的监测。Objective To verify an recombinase-isothermal amplification(RAA)assay to identify Staphylococcus aureus and Hemolytic streptococcus in areas of blood collection and production.Methods To prepare 10 cfu/ml-104 cfu/ml standard pathogen suspensions,add them to the rapid nucleic acid lysate,and extract the DNA by heating.To add RAA fluorescence detection reagent to the small Genchek fluorescence detector at 37℃,identify and amplify bacterial specific gene fragments,read real-time fluorescence detection values and determine results within 20 minutes.Eight standard strains were tested and their specificity was tested.The eight standard strains and 86 blood-supply environmental sanitation samples were tested using morphological-biochemical and RAA methods.Results The minimum detection limit of the two pathogenic bacteria by the RAA method is 102 cfu/ml,and there is no cross-reaction with other bacteria.The test results of the process hygiene samples during the blood collection and supply process are the same as the morphology-biochemical method.Conclusion The RAA technology can quickly,sensitively and specifically detect Staphylococcus aureus and Hemolytic streptococcus without isolation.The operation is fast and simple,and there is no special requirement for the experimenter.It can be used to monitor pathogenic bacteria in the blood supply environment.

关 键 词：致病菌检测 金黄色葡萄球菌 溶血性链球菌 重组酶 等温扩增 

分 类 号：R446.5[医药卫生—诊断学]