Miniaturized Single-Step Duplex CRISPR Diagnostic Platform for At-Home Molecular Testing of HPV16 and HPV18.

The early screening, precise diagnosis, and effective treatment of invasive cervical cancer necessitate at-home molecular testing of human papillomaviruses (HPVs). However, current HPV DNA tests cannot meet the need for an affordable, rapid, and accurate diagnosis using a streamlined workflow. Here, we present a miniaturized single-step duplex CRISPR diagnostic platform, termed SCOPEv2 (Streamlined CRISPR On Pod Evaluation platform, version 2), for rapid and highly sensitive at-home molecular testing of high-risk HPV16 and HPV18 for population screening of cervical cancer. Dual-target recombinase polymerase amplification (RPA) was initially incorporated with Cas12a/Cas13a cleavage reactions in a single-step reaction system. A miniaturized and low-cost dual-color wireless analysis device was further developed to execute the analysis workflow. SCOPEv2 can detect HPV16 and HPV18 with limits of detection of 2.5 copies/μL (5 copies/reaction) and 5 copies/μL (10 copies/reaction) in 30 min, respectively. The analysis results for 128 clinical cervicovaginal swab samples revealed 94.7% sensitivity and 100% specificity. SCOPEv2 demonstrates an easy-to-use workflow, low cost, high analytical performance, and superior clinical feasibility, which enable accurate and simultaneous point-of-care testing of HPV16 and HPV18.