快速等温扩增法在采供血机构沙门菌监测中的应用

A rapid recombinase-aided amplification assay for Salmonella bacteria detection in blood transfusion services

目的评价快速重组酶介导的等温扩增技术(RAA)在采供血机构沙门菌生物学监测中的应用。方法加热快速裂解肠炎沙门菌标准菌株提取DNA,采用沙门菌RAA荧光型检测试剂在小型Genchek荧光检测仪内37℃条件下识别并扩增沙门菌的特异基因片段,20 min内实时读取荧光检测值,根据扩增曲线判定结果。评估RAA法的检测灵敏度,同时采用RAA法检测13个标准菌株和5个实验室分离的沙门菌株验证其特异性。采集51份样本,分别接种肠炎沙门菌和鼠伤寒沙门菌标准菌株,以及无沙门菌污染的空白对照,营养琼脂平板培养48 h计数菌落数,同时采用国标GB 4789.4—2016的方法和RAA法进行沙门菌检测。结果RAA法在37℃20 min的条件下对肠炎沙门菌的最低检出限为1.9×10^2 CFU/mL,和非沙门菌无交叉反应。使用中消毒液和血液运输箱内壁沙门菌生物学的检测结果与国标法相同,Kappa值均为1。结论RAA技术可快速、灵敏、特异性地检测沙门菌,可应用于采供血机构沙门菌的生物学监测。

Objective To verify an isothermal real-time recombinase-aided amplification(RAA)assay to monitor Salmonella bacteria in blood transfusion services.Methods DNA was extracted from aliquots of standard Salmonella bacteria cultures of various concentrations.Specific gene fragments of Salmonella bacteria were detected and amplified in Genchek Fluorescence by using RAA assay of Salmonella at 37℃.Within 20 minutes,the values of fluorescence detection and amplification curve were analyzed to evaluate the sensitivity of this method.Assay specificity was validated by 13 standard bacteria of different species and 5 Salmonella bacteria reserved in the laboratory.The performance of the RAA assay was further evaluated with 51 samples.These samples were divided into three groups of Salmonella enteritidis inoculation,Salmonella typhimurium inoculation and blank,and were cultured on the nutrient agar plate for CFU counting after 48 h.Both RAA and GB4789.4-2016 assay were performed to analyze the Salmonella bacteria.Results The RAA assay could reliably detect Salmonella bacteria as few as 1.9×10^2 CFU/mL in pure culture at a constant temperature(37℃)within 20 minutes,and had no cross-reaction with other tested bacteria.The results of RAA assay were consistent with the GB method for samples taken from disinfectant in use and inner surface of blood transportation cooler box,and all the values of Kappa were 1.Conclusions RAA assay can provide rapid,sensitive and specific detection results for Salmonella bacteria,and can be used for Salmonella monitoring in blood transfusion services.