One-step reverse transcriptase-free miRNA detection system and its application for detection of gastrointestinal cancers

• Published in: Talanta (Oxford) Vol. 278; p. 126457
• Main Authors: Zhao, Guodong, Xue, Ying, Dai, Yanmiao, Zhou, Xiaojin, Li, Hui, Sheng, Guangsen, Xu, Hongwei, Chen, Ying
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2024
• Subjects: blood serum; cost effectiveness; detection limit; disease diagnosis; DNA-directed DNA polymerase; Gastrointestinal cancers; Gastrointestinal Neoplasms - diagnosis; Gastrointestinal Neoplasms - genetics; gastrointestinal system; genes; Humans; Limit of Detection; microRNA; MicroRNAs - analysis; MicroRNAs - blood; MicroRNAs - genetics; miRNA; OSMOS-qPCR; Real-Time Polymerase Chain Reaction; Reverse transcriptase-free; Taq Polymerase - metabolism; miRNA; Gastrointestinal cancers; OSMOS-qPCR; Reverse transcriptase-free
• ISSN: 0039-9140; 1873-3573; 1873-3573
• DOI: 10.1016/j.talanta.2024.126457

MicroRNAs (miRNAs) play pivotal roles in gene regulation and their dysregulation is implicated in various diseases, including cancer. Current methods for miRNA analysis often involve complex procedures and high costs, limiting their clinical utility. Therefore, there is a critical need for the development of simpler and more cost-effective miRNA detection techniques to enable early disease diagnosis. In this study, we introduce a novel one-enzyme for miRNA one-step detection method using Taq DNA polymerase, termed OSMOS-qPCR. We optimized the PCR buffer, PCR program, Taq DNA Polymerase concentrations and reverse PCR primer concentrations, resulted in a wide linear range from 100 fM to 0.001 fM (R2 > 0.98 for each miRNA), the detection limit for OSMOS-qPCR was 0.0025 fM. Furthermore, OSMOS-qPCR demonstrates excellent specificity to differentiation of less than 0.1 % nonspecific signal. Finally, we demonstrated the robust amplification efficiency, enabling the detection of trace amounts of cell-free miRNA in serum samples, and the excellent discrimination ability between gastrointestinal cancers and control subjects (AUC value = 1.0) if combined two miRNAs. The development of OSMOS-qPCR offering a simpler, cost-effective, and efficient detection method, has the potential to be non-invasive strategy for early detection of gastrointestinal cancers. [Display omitted] •First report the feasibility of single Taq DNA Polymerase for detection of miRNA.•One-step reverse transcriptase-free miRNA detection system with high specificity and low detection limit below 0.01 fM.•High accuracy for detecting cell-miRNA in serum for early detection of gastrointestinal cancers.