Rapid and multi-target genotyping of Helicobacter pylori with digital microfluidics

基因分型 幽门螺杆菌 数字聚合酶链反应 清脆的 重组酶聚合酶扩增 聚合酶链反应 生物 计算生物学 假阳性悖论 放大器 微流控 胃炎 基因 基因型 遗传学 计算机科学 纳米技术 材料科学 机器学习
作者
Liu Jinsong,Rongxin Fu,Shuailong Zhang,Jialu Hou,Hanbin Ma,Siyi Hu,Hang Li,Yanli Zhang,Weian Wang,Bokang Qiao,Baisheng Zang,Xun Min,Feng Zhang,Jie Du,Sheng-kai Yan
出处
期刊:Biosensors and Bioelectronics [Elsevier BV]
卷期号:256: 116282-116282 被引量:8
标识
DOI:10.1016/j.bios.2024.116282
摘要

Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.
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