新型发卡探针滚环扩增检测食品微生物的抗干扰机制及其方法构建

锁式探针滚环扩增是一种快速灵敏的等温核酸扩增技术，在货架期较短的生食和即食食品微生物现场快检中极具应用前景。但食品本底核酸含量可达微生物靶标的数千万倍，严重干扰锁式探针与靶标的杂交和成环，显著降低滚环扩增的特异性和灵敏度，使其难以规模化应用。因此，避免滚环扩增受食品复杂本底核酸的干扰是解决这一瓶颈问题的关键。本项目基于前期对含发卡DNA成环机理的研究，在锁式探针末端引入由单链DNA回折形成的发卡结构，减少滚环扩增中探针成环干扰，提高其靶标捕获特异性和成环率并阐明机制。通过研究发卡长度、序列和稳定性对发卡识别错配碱基的影响，获知高特异性发卡的关键特性；通过探明食品本底核酸和离子强度等因素对探针成环的影响，找出提高探针成环率的核心要素；构建新型发卡探针常温指数滚环扩增，实现食品中微生物的高特异高灵敏快速定性定量检测。本项目对滚环扩增技术在食品及其他复杂样品中的微生物快速检测找到一条新颖的路径。

Padlock probe-based rolling circle amplification (RCA) is a kind of rapid and sensitive isothermal amplification technology. Its application prospect in the on-site rapid analysis makes it ideal for the microorganism detection of raw food and instant food whose shelf lives are extremely short. While RCA technologies currently suffer from a challenging issue of background interference. The nucleic acids from food samples (background nucleic acids) are tens of millions of times as many as those from the microorganism genome, which seriously interferes with the target capture and cyclization of padlock probes. This interference significantly decreases the sensitivity and specificity of RCA, making RCA difficult to be applied on a large scale. Therefore, how to avoid the interference of large numbers of background nucleic acids on RCA is the key to solve this bottleneck problem. To this end, based on our preliminary researches on the circularization mechanism of DNA with hairpins, we innovatively propose to use a new type of padlock probe with terminal hairpins (called hairpin probe) to increase target capturing specificity and probe cyclization yield, and thus to significantly improve the anti-interference ability of RCA. Firstly, we will explore the effect of hairpin length, sequence and stability on the non-target mismatch recognition, and get the key structural characteristics of highly specific terminal hairpins. Secondly, we will investigate the influence of background nucleic acids and reaction conditions including ion strength on the cyclization of hairpin probe. Finally, a new exponential rolling circle amplification method based on hairpin probe at room temperature will be constructed to achieve highly specific and sensitive rapid detection of food-borne microorganisms. This project will find a novel path for the rapid detection of microorganisms in food and other complex samples by rolling circle amplification technology.