Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.