CBT-Cys click ligation coupled with pregnancy test strips for nucleic acid assay

Rolling circle amplification (RCA) is a powerful nucleic acid detection method widely used in molecular diagnosis. The circular template, which is an essential component of RCA, is typically generated via enzymatic ligation. However, enzymatic ligation has limitations such as high cost and strict reaction conditions, and nucleic acid detection usually relies on large instruments, which is difficult to promote in places with limited resources. Therefore, it is of great significance to construct a rapid, simple and efficient non-enzymatic ligation detection method.

Herein, a click chemistry-based method was developed for joining the sticky ends of DNA molecules in RCA. This approach leverages a 2-cyano-6-aminobenzothiazole (CBT) and cystine (Cys-Cys) modification at the nucleic acid termini (CBT-nucleic acid-Cys-Cys). When the HAV is present, a tightly packed circular DNA is formed. After the addition of glutathione, the disulfide bond is reduced, facilitating chemical linkage between CBT and Cys. Subsequently, RCA was initiated using phi29 polymerase. The RCA product and dsDNA magnetic conjugation sequence replacement enables the release of hCG-ssDNA2 for hepatitis A virus (HAV) quantification via pregnancy test strip (PTS) detection. The process was validated using mass spectrometry, gel electrophoresis and pregnancy test strip (T-line) signals. The method demonstrated a detection limit of 3.2 fM for HAV, with a linear range from 10.0 nM to 10.0 fM, showcasing its high sensitivity and specificity.

CBT-Cys click ligation has a high rate constant (13.82 M^-1 S^-1), offering better kinetic characteristics than the traditional enzyme-catalyzed ligation reaction, providing a milder and more flexible choice for DNA ligation methods. This efficient non-enzymatic ligation system exhibits promising potential for nucleic acid detection and clinical diagnostics, offering a promising approach for rapid, portable and cost-effective testing in future biomedical settings.