Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.面向生物安全应用的环境样本病原体检测与鉴定工作，因对特异性、灵敏度及检测时长的严苛要求而极具挑战性。本研究开发了一种可快速、高特异性且高灵敏地完成环境样本病原体鉴定的技术思路。靶标鉴定通过锁式探针（padlock probing）与邻近探针技术（proximity probing）实现，反应后的探针通过RCA（滚环扩增，rolling-circle amplification）进行扩增。各滚环扩增产物经荧光标记后，由专为高灵敏快速数字分析开发的专用仪器完成计数。本研究通过对野外释放的模拟生物战剂（细菌类包括大肠杆菌（Escherichia coli）、成团泛菌（Pantoea agglomerans），芽孢类包括萎缩芽孢杆菌（Bacillus atrophaeus））的鉴定，验证了该技术思路的可行性。