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hly A 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。
Catalytic transfer of genetic information from DNA to RNA is very important in life activities. The unconventional RNA transcription level may be related to the source of genetic diseases. At present, conventional methods for detection of RNA transcripts usually involve cumbersome preparative steps, or require sophisticated laboratory equipments. In this study, we presented a rapid, sensitive nano-detection platform for monitoring of RNA transcript levels. T7 RNA polymerase transcription reaction is employed as the example to test the feasibility of this method. In this design, in vitro synthesized RNA products can be hybridized to the FAM labeled single strand DNA (ssDNA) probes which can be adsorbed onto the graphene oxide (GO) surface. Using GO as the fluorescence switch, excellent capacity of the signal-on fluorescence platform for detection of RNA transcripts level is demonstrated. Transcription levels sensing with this nano-platform achieved a sensitivity of 5 pmol/L transcription template. It is antici- pated that current developed RNA transcript nano-detection mode has the potential to be an alternative to the conventional RNA transcript detection methods.
