<br>超灵敏多重miRNA一步实时RT-qPCR检测系统及其在食管癌血清中的应用,Biosensors and Bioelectronics

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超灵敏多重miRNA一步实时RT-qPCR检测系统及其在食管癌血清中的应用
Biosensors and Bioelectronics ( IF 10.7 ) Pub Date : 2023-12-15 , DOI: 10.1016/j.bios.2023.115927
Ying Xue ₁ , Kai Wang ₂ , Yunli Jiang ₃ , Yanmiao Dai ₄ , Xiaoyu Liu ₂ , Bing Pei ₅ , Hui Li ₃ , Hongwei Xu ₄ , Guodong Zhao ₆

Affiliation

MicroRNA (miRNA) 越来越被认为是早期疾病诊断和预后的有前景的生物标志物。因此，在生物研究和临床诊断中需要快速、稳健的多重 miRNA 检测方法至关重要。本研究介绍了一种新颖的多重 miRNA 检测方法，SMOS-qPCR（灵敏且多重的一步 RT-qPCR）。该方法将多重逆转录和基于 TaqMan 的 qPCR 集成到单个管中，在实时 PCR 系统上采用一步操作。我们研究了 Linker 3' 端磷酸化、Linker 浓度和探针浓度对 SMOS-qPCR 的影响，产生了从 1 fM 到 0.1 zM 的宽线性范围（每个 miRNA 的 R ≥ 0.99），超越了干细胞的能力-loop RT-qPCR 和 SYBR Green 一步法 RT-qPCR。该方法在区分成熟miRNA和miRNA前体方面表现出优异的性能，并成功检测单管中的4个miRNA，且无交叉干扰。其高特异性能够精确区分小于 1% 的非特异性信号。最后，我们证明了 SMOS-qPCR 系统在检测血清样本中的循环 miRNA 方面的有效性，区分食管癌和具有高 AUC 值 (>0.940) 的健康个体。总之，所提出的 SMOS-qPCR 系统为未来临床应用中的 miRNA 分析提供了一种简单且有前景的方法。

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An ultrasensitive and multiplexed miRNA one-step real time RT-qPCR detection system and its application in esophageal cancer serum

MicroRNAs (miRNAs) are increasingly recognized as promising biomarkers for early disease diagnosis and prognosis. Therefore, the need for rapid, robust methods for multiplex miRNA detection in biological research and clinical diagnosis is crucial. This study introduces a novel multiplex miRNA detection method, SMOS-qPCR (Sensitive and Multiplexed One-Step RT-qPCR). The method integrates multiplexed reverse transcription and TaqMan-based qPCR into a single tube, employing a one-step operation on a real-time PCR system. We investigated the effect of 3′ end phosphorylation of the Linker, Linker concentration and probe concentration on the SMOS-qPCR, resulted in a wide linear range from 1 fM to 0.1 zM (R ≥ 0.99 for each miRNA), surpassing the capabilities of stem-loop RT-qPCR and SYBR Green One-step RT-qPCR. The method showed excellent performance in distinguishing mature miRNA from miRNA precursor, and successfully detected four miRNAs in a single tube without cross-interference. Its high specificity enables precise differentiation of less than 1% nonspecific signal. Finally, we demonstrated the effectiveness of the SMOS-qPCR system in detecting circulating miRNAs in serum samples, distinguishing between esophageal cancers and health individuals with high AUC values (>0.940). In conclusion, the proposed SMOS-qPCR system offers a straightforward and promising approach for miRNA profiling in future clinical applications.

更新日期：2023-12-15

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