联营
熔化曲线分析
计算生物学
化学
多路复用
RNA提取
色谱法
实时聚合酶链反应
复制
再现性
逆转录聚合酶链式反应
生物系统
核酸
逆转录酶
聚合酶链反应
核糖核酸
生物
计算机科学
统计
生物信息学
信使核糖核酸
数学
人工智能
生物化学
基因
作者
Xinyu Zhuang,Zibin Zhao,Xianzhen Feng,Grace Lui,Daniel Chan,Shui Shan Lee,I‐Ming Hsing
标识
DOI:10.1021/acs.analchem.3c00885
摘要
Pooling multiple samples prior to real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to minimize expenses and boost test throughput during the COVID-19 pandemic. Nevertheless, the traditional pooling approach cannot be effectively deployed in high-prevalence settings due to the need for secondary tests in the case of a positive pool. In this study, we present a pooling test platform with high adaptability and simplicity that allows sample-specific detection of multiple-tagged samples in a single run without the need for retesting. This was accomplished by labeling distinct samples with predefined ID-Primers and identifying tagged pooled samples using one-step RT-PCR followed by melting curve analysis with rationally designed universal fluorescence- and quencher-tagged oligo probes. Using magnetic beads (MBs), nucleic acid targets from different individuals can be tagged and extracted concurrently and then pooled before RT, eliminating the need for extra RNA extraction and separate RT and enzyme digestion steps in the recently developed barcoding strategies. Pools of six samples (positive and negative) were successfully identified by melting temperature values under two fluorescent channels, with a detection sensitivity of 5 copies/μL. We validated the reproducibility of this assay by running it on 40 clinical samples with a hypothetical infection rate of 15%. In addition, to aid the scenario of large-scale pooling tests, we constructed a melting curve autoreadout system (MCARS) for statistical analysis of melting curve plots to eliminate error-prone manual result readout. Our results suggest that this strategy could be a simple and adaptable tool for alleviating existing bottlenecks in diagnostic pooling testing.
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