实时荧光跨越式滚环等温扩增技术检测食品中的志贺氏菌  

Combining Real-time Fluorescence with Saltatory Rolling Circle Amplification for Detection of Shigella in Food

作　　者：王立娟 郭威 张先舟[1] 马晓燕[1] 张伟[1,2,3] WANG Li-juan;GUO Wei;ZHANG Xian-zhou;MA Xiao-yan;ZHANG Wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,Hebei,China;College of Life Sciences,Hebei Agricultural University,Baoding 071001,Hebei,China;College of Science and Technology,Hebei Agricultural University,Cangzhou 061100,Hebei,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]河北农业大学生命科学学院,河北保定071001 [3]河北农业大学理工学院,河北沧州061100

出　　处：《食品研究与开发》2021年第14期125-131,共7页Food Research and Development

基　　金：国家自然科学基金(31371772);河北省自然科学基金重点项目(C2019204342)。

摘　　要：该研究建立一种实时荧光跨越式滚环等温扩增(real-time fluorescence saltatory rolling circle amplification,RFSRCA)技术快速检测志贺氏菌(Shigella)。该方法以志贺氏菌的ipaH基因设计引物,使用32株不同菌株对RF-SRCA方法的特异性进行分析,根据实时荧光曲线法对RF-SRCA方法的灵敏度和检出限进行判定,并使用该方法对60份食品样品进行检测。结果表明:13株志贺氏菌菌株呈阳性结果,19株非志贺氏菌菌株呈阴性结果,说明该方法特异性良好;RF-SRCA的灵敏度为5.97×10^(0)fg/μL,比普通SRCA方法高10倍;其在人工污染牛奶样品中的检出限为8.6×10^(0)CFU/mL,比普通SRCA方法检出限低10倍;60份食品样品中阳性样品数为2份,其检出率与SRCA方法一致,为3.33%。综上,RF-SRCA方法在检测志贺氏菌方面操作简单,特异性强,灵敏度高,能够实现快速检测。A method combining real-time fluorescence detection with saltatory rolling circle amplification(RFSRCA)was established in this study for rapid Shigella detection. Primers for this method were designed complementary to Shigella’s ipaH gene. A total of 32 different strains were assessed to analyze specificity. Sensitivity and detection limit were determined according to the real-time fluorescence curve method. RF-SRCA was used to test 60 food samples for Shigella. Thirteen Shigella strains gave positive results and negative results were obtained from 19 non-Shigella strains,indicated that the method confers good specificity. The sensitivity of RFSRCA was 5.97×10^(0) fg/μL,10 times higher than that of SRCA. The detection limit in artificially contaminated milk samples using RF-SRCA was 8.6×10^(0) CFU/mL,which was 10 times lower than the limit using SRCA. Two positive samples were detected from among 60 food samples. The detection rate of RF-SRCA was 3.33%,consistent with that of SRCA. In summary,RF-SRCA offered ease of operation,strong specificity,high sensitivity,and a low detection limit when used for rapid detection of Shigella.

关 键 词：志贺氏菌 实时荧光跨越式滚环等温扩增技术 ipaH 检测 牛奶 

分 类 号：R155.5[医药卫生—营养与食品卫生学]