烈性噬菌体结合生物膜干涉技术的单增李斯特菌实时无标记快速检测

Listeria monocytogenes (LM), which causes food poisoning and listeriosis with a high mortality of 30%, is a severe threat to human. The rapid and reliable detection of LM is critical for the effective prevention and control of outbreaks of food poisoning and listeriosis. Biolayer interferometry (BLI) biosensor and virulent bacteriophage were used to develop a rapid detection method for LM. Virulent bacteriophages were isolated from raw sewage using LM serotypes 1/2a, 1/2b and 4b strains. The host range of these bacteriophages was determined by spot-testing. Bacteriophages lytic against 1/2a, 1/2b and 4b serotype strains with no lysis against other tested bacteria strains were selected for further evaluation. The influence law of the composition ratio of self-assembled monolayer on the immobilization of bacteriophage was revealed, and the appropriate modification method for biosensor surface was determined. The immobilization efficiency of selected bacteriophages on BLI biosensor surface was investigated and bacteriophages with high efficiency were selected for the analysis of detection sensitivity and specificity. The bacteriophage with the highest sensitivity and specificity was used to develop the rapid detection method. This method allows for real-time, label-free and rapid detection of LM and also can distinguish between dead and living cells. The study is expected to open up a new research field for rapid detection of LM. The developed method and the revealed law would give an important guiding significance for development of rapid detection method for other foodborne pathogens.

单增李斯特菌（Listeria monocytogenes, LM）能引起食物中毒和李斯特菌病，病死率高达30%。快速准确检测LM是预防和控制食物中毒及李斯特菌病的关键。项目以烈性噬菌体结合生物膜干涉（biolayer interferometry，BLI）技术探索LM快速检测新途径。先以目标LM菌为宿主菌分离LM烈性噬菌体，经谱系分析选出有高度特异性的LM噬菌体；然后揭示自组装单分子层的组分配比对噬菌体固定化的影响规律，确定合适的传感器表面修饰方法；而后考察噬菌体在传感器表面的固定化效果及检测的灵敏度和特异性，选出适合做BLI生物传感器生物识别元件的LM烈性噬菌体，并最终建立基于烈性噬菌体和BLI技术的LM快速检测方法。方法可实现LM的实时无标记快速检测，并可区分死活菌。项目有望为LM快速检测开辟一个新方向，所做的探索及揭示的规律对于LM以外的其他食源性致病菌的快速检测也有重要指导意义。

食源性疾病是世界性的公共卫生问题，食源性致病菌是引发食源性疾病的最主要因素。单增李斯特菌能引起食物中毒和李斯特菌病，病死率高达30%。沙门氏菌是全球腹泻病的四大病因之一，在食源性致病菌中排名第一。对它们的快速准确检测是预防和控制由此引起的食物中毒及相关疾病的关键。传统检测方法步骤多、操作繁琐、耗时长，开发更加简单、快速、准确的新型检测方法是迫切需要的。项目利用新一代的实时免标记快速检测技术即生物膜干涉（biolayer interferometry，BLI）技术探索单增李斯特菌和沙门氏菌的检测新方法，主要研究内容有（1）烈性噬菌体结合BLI技术的单增李斯特菌快速检测，具体包括单增李斯特菌烈性噬菌体在BLI生物传感器上的固定化、噬菌体的生物素化、检测方法的灵敏度分析；（2）抗体结合BLI技术的单增李斯特菌快速检测，具体包括醋酸-醋酸钠缓冲溶液最佳pH值的确定、抗体固定化时最佳抗体浓度的确定、检测方法的灵敏度分析；（3）抗体结合BLI技术的沙门氏菌快速检测，具体包括缓冲溶液最佳pH值的确定、抗体固定化时最佳抗体浓度的确定、方法的灵敏度分析、检出限确定、特异性分析、以及实际食品样品中沙门氏菌的检测。项目最终成功构建了基于BLI技术的快速检测新方法，实现了单增李斯特菌和沙门氏菌的实时无标记快速检测。以噬菌体和抗体为识别元件检测单增李斯特菌时，检出限分别为9.74×10^4（结合时间为20 min）和3.66×10^4 CFU/mL（结合时间为6 min）；以抗体为识别元件检测沙门氏菌时，检出限为5.6×10^5 CFU/mL（结合时间为5 min）。除检测方法的研究外，项目还针对单增李斯特菌等重要食源性致病菌研制了新的通用增菌培养基。项目为食源性致病菌快速检测开辟了一个新方向，所做的探索对于单增李斯特菌和沙门氏菌以外的其他食源性致病菌的快速检测也有重要指导意义。