不对称PCR结合滚环扩增信号放大技术检测牛肉中单增李斯特菌  

Detection of Listeria monocytogenes in beef by asymmetric PCR combined with rolling ring amplification

作　　者：龙慧[1] 钟淙[1] 帅淑芬 龙永艳[1] 王伟[1] 吴葵 吴鑫 LONG Hui;ZHONG Cong;SHUAI Shufen;LONG Yongyan;WANG Wei;WU Kui;Wu Xin(Nanchang Center for Disease Control and Prevention,Nanchang Jiangxi,330038,China;Jiangxi General Institute of Testing and Certification Food Testing Institute)

机构地区：[1]南昌市疾病预防控制中心,江西南昌330038 [2]江西省检验检测认证总院食品检验检测研究院

出　　处：《质量安全与检验检测》2023年第1期89-94,共6页QUALITY SAFETY INSPECTION AND TESTING

基　　金：南昌市科技支撑计划项目(洪科字〔2021〕129号);江西省检验检测认证总院科研项目(ZYK202212)。

摘　　要：建立一种不对称聚合酶链式反应结合滚环扩增信号放大(Rolling circle amplification,RCA)的技术,对牛肉中单增李斯特菌进行快速灵敏检测。通过改变上下游引物的浓度,扩增得到一条带有通用序列的单链DNA,进而引发RCA反应,产生大量的G-四链体序列,硫黄素T嵌入G-四链体序列中产生荧光信号,从而实现对单增李斯特菌的检测。在最优的条件下,本研究所建立的方法对单增李斯特菌在PBS中的检测限为3.6×101 CFU/mL;在加标的牛肉中,其检测限达到了3.6×102 CFU/g。本研究所建立的方法可实现单增李斯特菌的快速检测,通过改变特异性的引物,该方法也可用于其他食源性致病菌的检测,具有较高的推广应用价值。An asymmetric polymerase chain reaction-combined rolling cycle amplification(aPCR-RCA)was established to conduct rapid and sensitive detection to the Listeria monocytogenes in beef.A single-strand DNA is obtained by amplification in the modified concentration of primers.Then the single-strand DNA induces RCA reaction to produce G-quadruplex sequence.The embedding of thioflavin T into the G-quadruplex sequence generates fluorescence signal,thus realizing the detection of Listeria monocytogenes.Under the optimal condition,detection limit of the established method to Listeria monocytogenes in PBS is 3.6×101 CFU/mL.In the labelling beef,detection limit of this method can reach 3.6×102 CFU/g.The established method can achieve rapid test to Listeria monocytogenes.Additionally,this method can be applied to detect other food-derived pathogens through changing the specific primers and has high application value.

关 键 词：单增李斯特菌 牛肉 滚环扩增信号放大 检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学]