Abstract：
Point of care testing (POCT) of nucleic acid (NA) contributes to the timely disease diagnosis, like bacteria and virus screening in households or resource-constrained areas, but its development has always been stagnant. Herein, we proposed an exonuclease III cascaded with CRISPR/Cas12a (Exo-III/Cas12a) amplification strategy and constructed a smartphone-based portable fluorescence detector (SPFD) to repurpose the commercial alpha-fetoprotein (AFP) strip for the ultrasensitive and hand-held detection of NA samples. In detail, the target-initiated-Exo-III/Cas12a strategy realizes the signal amplification and liberates AFP from magnetic beads through the trans-cleavages of activated Cas12a toward the AFP aptamer. After magnetic separation and migration, the fluorescence signals of the test (FT) and control (FC) lines on the AFP strip were digitally output by the SPFD, and the FT/FC was employed for the quantitative analysis to minimize external disturbances and improve accuracy. We experimentally assessed the universe applicability of the proposed NA-POCT platform toward miRNA-155, 16S rRNA of Staphylococcus aureus, and ORF1a/b RNA of Covid-19 pseudovirus, achieving favorable detection limits of 42 aM, 18 CFU/mL, and 87 copies/μL, respectively. Moreover, its simplicity, universality, and admirable detection performance demonstrate a great potential in the aspect of rapidly transforming the existing POCT devices for multiple new applications at the time of need.
摘要：
核酸（NA）的即时检测（POCT）有助于及时诊断疾病，比如在家庭或资源有限的地区进行细菌和病毒筛查，但是它的发展一直停滞不前。在这里，我们提出了一种与CRISPR/Cas12a(Exo-III/Cas12a)级联的外切核酸酶III扩增策略,并构建了一个基于智能手机的便携式荧光检测器(SPFD),以重新利用商用甲胎蛋白(AFP)条进行NA样品的超灵敏和手持检测.详细来说,目标启动的Exo-III/Cas12a策略实现了信号放大，并通过激活的Cas12a的反式切割将AFP从磁珠释放到AFP适体。磁选和迁移后，AFP条上的测试(FT)和对照(FC)线的荧光信号由SPFD数字输出，并采用FT/FC进行定量分析，以最大程度地减少外部干扰并提高准确性。我们通过实验评估了所提出的NA-POCT平台对金黄色葡萄球菌的miRNA-155，16SrRNA，和Covid-19假病毒的ORF1a/bRNA，达到42aM的有利检测限，18CFU/mL,和87个拷贝/μL，分别。此外，它的简单性，普遍性,和令人钦佩的检测性能在快速转变现有的POCT设备在需要的时候为多个新的应用方面显示出巨大的潜力。
