激光生物学报, 2017, 26 (6): 527, 网络出版: 2018-01-10  

基于滚环扩增技术的纸基显色传感器用于致病菌快速检测

Rapid and Isothermal Paper-based Gene-sensing of Viable Pathogens with Rolling Circle Amplification
作者单位
华南师范大学生物光子学研究院, 激光生命科学研究所教育部重点实验室, 广东 广州 510631
摘要
近年来, 食源性致病菌在全球范围内大面积传播, 给人类的生存健康与发展带来了巨大的威胁。因此为了保证食品的安全, 快速的、高特异性且高灵敏地检测食品致病菌方法已经成为科学研究的热点话题。在本研究中, 我们构建了一种基于滚环扩增技术的纸基显色传感器用于可视化检测致病菌的检测平台。通过滚环扩增对单增李斯特菌的hlyA mRNA进行高效特异性扩增, 扩增后的单链产物经过Hhal酶进行特异位点酶切后, 形成与锁式探针长度相同的片段。将酶切后的单链产物不经过预变性杂交, 可直接进行试纸条检测。在优化的实验条件下, 可以检测到100 pg/μL总RNA。整个过程实验包括连接, 扩增, 酶切, 检测等反应均可在恒温条件下进行, 且可在几个小时内完成。这种方法适用于快速的现场检测, 此外, 这项研究强调了纸基显色诊断平台的潜在价值和应用前景。
Abstract
In economic terms, the microbiological safety of food is a serious concern of consumers and the food industry owing to the significant public health and global economic impact of foodborne diseases. Thus, specific and sensitive detection of pathogenic bacteria is required to ensure food safety. In this study, we constructed a novel paper-based nucleic acid diagnostics for viable pathogenic bacteria detection with naked eyes. An effective isothermal amplification method was described to amplify the hlyA mRNA gene of Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test without pre-hybridization. When the DNA products migrated along the platform by capillary action, the gold-nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 100 pg/μL total RNA could be detected. The whole assay process, including ligation, amplification, enzyme digestion, and detection, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens. Furthermore, this study highlights the potential of the paper-based nucleic acid diagnostics platform for cost- and labor-effective applications in the detection of pathogenic bacteria.

黄梦琪, 刘芳, 周小明. 基于滚环扩增技术的纸基显色传感器用于致病菌快速检测[J]. 激光生物学报, 2017, 26(6): 527. HUANG Mengqi, LIU Fang, ZHOU Xiaoming. Rapid and Isothermal Paper-based Gene-sensing of Viable Pathogens with Rolling Circle Amplification[J]. Acta Laser Biology Sinica, 2017, 26(6): 527.

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