基于磁分离与透射-散射式浊度法的致病菌现场核酸定量检测一体化研究

Foodborne pathogenic bacteria is an important factor for endangering food safety. Due to the problems of low sensitivity, low automation and cross-pollution, the current detection methods of pathogenic bacteria have been more and more unable to meet the requirements of food safety testing. This project presents a field-based and integrated nucleic acid quantitative testing method for pathogenic pathogen by means of magnetic separation and transmitter-scattering turbidometry, the major studying contents including: (1) optimizing the condition of nucleic acid extraction from pathogenic bacteria using magnetic beads in food, the loop-mediated isothermal amplification using real-time turbidometry and automatic parameter. (2) constructing the model of portable and closed cartridge, high efficiency magnetic separation, constant temperature control and transmission-scattering multi-channel turbidity analysis, and so on. (3) studying automatic control circuit, algorithm and human-computer interaction interface for cartridge. (4) evaluating the performance and biochemical indicators for integrated quantitative detection of nucleic acid in pathogenic bacteria. The above integrated detection method has advantages of more convenience, higher detection sensitivity and efficiency, and the studied cartridge has the characteristics of safe and reliable, easy to make and use, higher integration, easy to expand, and so on, thus ensuring the simplicity, accuracy and timeliness for on-site integrated quantitative or multiple detection of nucleic acid in foodborne pathogenic bacteria, also providing new ideas for food safety and precise medical.

食源性致病菌是危害食品安全的重要因素。由于存在灵敏度不高、自动化程度低与交叉污染等问题，现行致病菌检测方法已越来越不能满足食品安全检测的要求。本项目提出了一种基于磁分离与透射-散射式浊度法的致病菌现场核酸定量检测一体化方法，主要研究内容为:(1)食品中致病菌核酸磁珠法提取最佳条件、LAMP实时浊度检测体系最佳条件与自动控制参数的优化;(2)构建便携式封闭卡盒、高效磁分离、恒温控制与透射-散射式多通道浊度分析等模型;(3)研究自动化控制电路、算法及人机交互界面;(4)评价食源性致病菌核酸定量检测一体化性能及生化指标。上述一体化检测方法具有操作更加便利、灵敏度及检测效率更高的优点，所研究的卡盒具有安全可靠、易制作与使用、集成度更高与易于扩展等特点，确保了现场食源性致病菌一体化核酸定量或多重检测的简易性、准确度及时效性，为食品安全和精准医学提供了新思路。

食源性致病菌是引起食品安全问题的重要原因，也是全球食品安全的核心问题。由于存在灵敏度不高、自动化程度低与交叉污染等问题，近年来，传统检测技术暴露出检测耗时长效率低等，免疫学方法总体灵敏度不高等问题，越来越不能满足现代食品工业对安全检测的要求以及公共安全事件应急处理快速反应的需要。本项目提出了一种基于磁分离与透射-散射式浊度法的致病菌现场核酸定量检测一体化方法，主要研究内容包括:(1) 基于磁珠法核酸提取与LAMP浊度法检测PAE自动化方法建立;(2) 设计了基于卡盒的自动化模型、卡盒驱动模型；研制了磁分离、超声、光电、移液、旋转运动等模块;(3) 开发了卡盒的自动化控制软硬件系统，实现了温度控制、运动控制、光电检测、外部通信等功能；(4)评价了PAE一体化检测器件性能及生化指标。最终研制了一种快速、便捷、高灵敏、高特异性的食源性致病菌核酸信息定量检测一体化新系统和铜绿假单胞菌（PAE）检测新方法。针对PAE检验，该系统采用的透射和散射法均可以实现检测，检测时间约60-90分钟，灵敏度为2.58 fg/μL，特异性100%；温度控制准确度±0.5℃。此外，散射法检测时间要快于且优于透射法，浊度最佳的检测波长在650nm。所研究系统具有操作便利、灵敏度及检测效率高的优点，所研究卡盒具有安全可靠、易制作与使用、集成度更高与易于扩展等特点，确保了现场食源性致病菌一体化核酸定量或多重检测的简易性、准确度及时效性，为食品安全和精准医学提供了新思路。