肺癌的早期诊断是肺癌防治的关键。研究显示肺癌患者血清中存在特异性变化的miRNA可作为一种新生物标志物，对肺癌的早期诊断、分期及预后评估具有重要价值。但是，现阶段miRNA检测技术存在成本高、操作复杂等缺陷，阻碍了其临床应用。本项目拟应用单链特异性核酸酶，开发一种低成本、操作简便、可高通量检测miRNA的新技术。通过核酸酶能够特异性识别并切割未配对单链核酸和碱基错配双链核酸的能力，检查核酸探针(酰胺反应连接至96孔酶标板)与目标miRNA(提取自血清)杂交中发生的错配和部分互补；然后利用结合到核酸探针上的辣根过氧化物酶与化学发光底物发生酶促反应产生荧光，快速放大检测信号；根据荧光强度确定目标miRNA含量。本项目拟用这一技术检测非小细胞肺癌患者血清样本中miRNA标志物，确定技术的最佳应用条件，并评估其灵敏度、特异性、实用性以及在肺癌早期诊断中的价值；为临床癌症(肺癌)的诊断提供新方法。

Early diagnosis is important for the prevention and treatment of lung cancer. Studies have shown that specific changes in the serum miRNA of lung cancer patients can be used as a novel biomarker, which has important value in the early diagnosis, staging, and prognosis evaluation of lung cancer. However, there are many defects in current miRNA detection techniques, such as high cost and complexity of operation, which hinder their clinical application. This project intends to develop a novel high-throughput miRNA detection technique with low-cost and easy to operate by applying a single-strand-specific nuclease (SSSN). Depending on the ability of the nuclease to specifically recognize and cleave unpaired single-stranded nucleotides and mismatched double-stranded nucleotides, we can check partially complementary and mismatched nucleotides in the hybridization between specific nucleic acid probes (fixed onto a 96-well enzyme-linked immunosorbent assay (ELISA) plate via the amide reaction) and target miRNA (extracted from the serum samples). Afterwards, the enzymatic reaction between horseradish peroxidase conjugated to the nucleic acid probes and a chemiluminescent substrate allows quick amplification of detectable fluorescence signals. The concentration of target miRNA can subsequently be determined from the fluorescence intensity. In this project, we also aim to utilize this novel technique to detect the serum miRNA biomarkers in the samples of nonsmall cell lung cancer patients and define its optimal application conditions, which assesses the sensitivity, the specificity, the practicability and the value of the technique in the early diagnosis of lung cancer. This project would provide a new method of clinical cancer diagnosis (e.g. lung cancer) for the future.