新型肽核酸-核酸适体三链探针构建及其在多菌复杂食品基质中瑞士乳杆菌的快速检测应用

益生菌是一类能够对宿主健康产生有益作用的活的微生物，其中瑞士乳杆菌凭借其优良的加工和益生特性在食品发酵工业中占据重要地位。为了保证益生菌类食品充分发挥益生作用，产品的活菌种类及其在保质期内的活菌数是两大质量关键，但目前用于益生菌的判别方法存在耗时长和成本高等问题。前期研究中，申请人利用PNA-DNA杂合探针和核酸适体实现了多种生物靶标的快速检测方法。本课题拟以从中国本土酸奶中分离到的两种具有显著益生功能的瑞士乳杆菌为检测对象，首先利用non-SELEX技术筛选通用型核酸适体，将核酸适体与PNA结合形成PNA-适体三链探针，并基于该探针构建特异性响应的多色比色型生物传感方法，进一步利用探针与纳米材料之间的相互作用实现多色信号输出，仅通过肉眼观察不同颜色变化即可实现多菌复杂食品基质中靶菌株的快速、准确、一体化检测及活性评估。本项目将为开发复杂基质中益生菌的快速鉴别及活性评估方法提供新策略。

Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. Lactobacillus helveticus plays an important role in food industry due to its superior biological characteristics. In alignment with the definition of probiotics and the requirements of probiotics to be effective, the species, strain names and minimum viable cell count in colony-forming units (CFUs) at expiration date on labels are key quality factors. However, the methods currently applied for probiotics identification are time-consuming and expensive. In previous studies, applicant proposed several detection methods for a variety of biomarkers by using PNA-DNA hybrid probes and aptamer. Herein, we choose two L. helveticus strains from Chinese yogurt, screen specific aptamer based on novel non-SELEX technology and bind with PNA to form PNA-aptamer triplex probe. The interactions between PNA-aptamer triplex probe and nanomaterials are further explored to design label-free multi-colorimetric aptasensors for probiotic identification and bacterial enumeration. This project is considered to provide new strategies for rapid, specific identification of probiotics in multi-bacteria complex food matrix.