基于末端转移酶核酸信号放大技术的沙门氏菌比色适配体传感器  

Aptasensor Based on Terminal Deoxynucleotidyl Transferase-Mediated Signal Amplification for Salmonella Typhimurium Detection

作　　者：张倩雯 陈谦 卞晓军[1,2,3] 颜娟 ZHANG Qian-Wen;CHEN Qian;BIAN Xiao-Jun;YAN Juan(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation,Shanghai 201306,China;Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation(Shanghai),Ministry of Agriculture,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海水产品加工及贮藏工程技术研究中心,上海201306 [3]农业部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306

出　　处：《分析化学》2021年第8期1289-1299,共11页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21775102);上海市自然科学基金项目(No.20ZR1424100)资助。

摘　　要：以鼠伤寒沙门氏菌(S.typhimurium)作为目标菌,制备了基于醛基化磁珠(Aldehyde magnetic beads,Mbs)、酶切技术和末端转移酶(Terminal deoxynucleotidyl transferase,TdT)介导的信号放大技术可用于食品安全的比色适配体(Aptamer,Apt)传感器(Aptasensor)。首先利用S.typhimurium适配体及与其序列互补的捕获探针(Capture probe,CP)制备了Mbs@dsDNA(Double-stranded DNA,dsDNA)杂交复合物,当待测物中含有S.typhimurium时,磁珠表面的适配体序列与其发生特异性结合,从磁珠表面释放,并通过磁响应去除;此时,磁珠表面的捕获探针自身回折,部分碱基互补配对并形成酶切识别区。限制性内切酶Ⅰ(EcoRⅠ)酶切后获得3′-OH端,在末端转移酶的作用下催化脱氧核苷酸(dNTPs)结合到3′-OH端,获得长单链DNA(Single stranded DNA,ssDNA)产物。在此过程中,信号报告分子不断嵌入ssDNA中,从而获得增强的检测信号。当待测物中不含S.typhimurium时,由于捕获探针与适配体3′-OH端均被磷酸基团封闭,并且无酶切过程产生的3′-OH端,因此无法引发TdT信号放大反应。比色适配体传感器对101-105 CFU/mL浓度范围内的S.typhimurium具有良好的响应性能,检出限低至21 CFU/mL,具有较高的灵敏度和特异性。此传感器检测牛奶样品中S.typhimurium的回收率为92.2%-112.7%(RSD<3%),在食品安全和污染物检测及监管方面具有潜在的应用价值。Salmonella typhimurium(S.typhimurium)is one of the most widespread pathogens causing foodborne diseases.In this work,S.typhimurium was used as the model bacteria.An aptasensor based on aldehyde magnetic beads(Mbs),enzyme digestion and terminal deoxynucleotidyl transferase-mediated signal amplification technique for simple colorimetric detection of S.typhimurium was developed.The Mbs@dsDNA hybrid complex was first prepared using the capture probe(CP)and aptamer.In the presence of S.typhimurium,the aptamer specifically bound to it,and released from the Mbs,which was then removed by magnetic separation.Subsequently,CP on Mbs folded back on itself owing to partially complementary bases and formed enzyme digestion recognition sites.3′-OH terminal ends were obtained by EcoR I enzyme digestion.Deoxynucleotides were catalyzed to the 3′-OH end under the action of terminal deoxynucleotidyl transferase(TdT)to obtain products of single-stranded DNA(ssDNA).In the process,signal reporter molecules were embedded in the chain of ssDNA,thus obtaining amplified signals.In the absence of S.typhimurium,TdT signal amplification reaction could not be initiated because no released aptamer and blocked 3′-OH end CP.The aptasensor had a good response performance to S.typhimurium in the concentration range of 101-105 CFU/mL,and the detection limit was as low as 21 CFU/mL,showing high sensitivity and specificity.Moreover,the recovery of S.typhimurium in milk samples was 92.2%-112.7%(RSD<3%).The aptasensor showed potential application prospect in the food safety detection and supervision of other foodborne pathogenic microorganisms and other pollutants.

关 键 词：生物传感器 适配体 信号放大技术 食源性疾病 食品安全 

分 类 号：TS207.4[轻工技术与工程—食品科学]