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投稿时间：2020-09-27
投稿时间：2020-09-27
中文摘要: 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5.97×100fg/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8.6×100CFU/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。
中文关键词: 志贺氏菌 实时荧光跨越式滚环等温扩增技术 ipaH 检测 牛奶
Abstract: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 5.97×100fg/μL，10 times higher than that of SRCA.The detection limit in artificially contaminated milk samples using RF-SRCA was 8.6×100CFU/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.
keywords: Shigella real-time fluorescent saltatory rolling circle amplification（RF-SRCA） ipaH detection method milk
文章编号：202114020 中图分类号： 文献标志码：
基金项目:国家自然科学基金（31371772）；河北省自然科学基金重点项目（C2019204342）
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