CRISPR/Cas12-based signal-on lateral flow immunoassay for ultra-sensitive, selective, and rapid detection of human papillomavirus DNA in clinical samples

The trans-cleavage capability of CRISPR/Cas12a presents significant opportunities for the precise detection of double-stranded DNA (dsDNA). However, this potential has not been fully utilized in the context of lateral flow devices. This study introduced a novel, highly sensitive lateral flow assay that leverages the functionalities of CRISPR/Cas12a in conjunction with DNA probes to detect human papillomavirus (HPV) DNA, a primary etiological factor in cervical cancer, directly from clinical samples. The assay employed a DNA capture probe with specific sequences to construct both the control line using 40 thymine bases (T40) single-stranded DNA (ssDNA) and the test line utilizing 40 cytocine bases (C40) ssDNA, thereby effectively monitoring the presence of target DNA. In the mixed master reaction, CRISPR/Cas12a was combined with Biotin-GGGGGGGGGGAAAAAAAAAA (G10A10) ssDNA and introduced to a sample containing the HPV DNA. This process initiated a trans-cleavage mechanism, resulting in the conversion of Biotin-G10A10 ssDNA into Biotin-G10 ssDNA. By incorporating streptavidin-conjugated gold nanoparticles on the conjugate pad, the assay captured both Biotin-G10 ssDNA and any unreacted Biotin-G10A10 ssDNA at the test line and control line, respectively, leading to a distinct colorimetric appearance. Under optimized conditions, the assay demonstrated a detection limit of 0.2 copies/μL and exhibited excellent selectivity for HPV16 in clinical samples, correlating favorably with results obtained through gel electrophoresis. This innovative system not only underscores the considerable potential for rapid, sensitive, and specific detection of HPV DNA, but it also serves as a promising prototype for further advancements in nucleic acid-based detection methodologies. • First “signal on” LFA to leverage the CRISPR/Cas12a trans-cleavage selectivity. • First CRISPR/Cas12a LFA to use ssDNA capture probe on both lines without antibodies. • High specificity and sensitivity with the low limit of detection of 0.2 copies/μL. • Promising results for clinical sample analysis, as verified by gel electrophoresis.