Rapid and Visual Detection of the tet(X4) Gene Using LAMP-CRISPR/Cas12a and RPA-CRISPR/Cas12a

The increasing prevalence of the plasmid-mediated tigecycline resistance gene tet(X4) has severely limited clinical treatment options. Isothermal amplification techniques, which do not require thermal cycling equipment, are designed for rapid clinical detection. Therefore, this study aims to establish and comparatively evaluate two rapid and visual detection technologies for tet(X4): Recombinase polymerase amplification (RPA)-CRISPR/Cas12a and Loop-mediated isothermal amplification (LAMP)-CRISPR/Cas12a nucleic acid detection system. The two methods were compared based on amplification temperature, amplification time, sensitivity, and specificity. The results demonstrated that the LAMP amplification required a reaction time of 50 min, with an optimal temperature of 65°C and a detection limit of 5 copies. The amplification products were further detected using CRISPR/Cas12a, and optimal results were obtained after a 30 min reaction at 35°C. In comparison, RPA exhibited a shorter reaction duration (30 min) and a lower reaction temperature (37°C), while maintaining the same detection limit of 5 copies. Detection using the CRISPR/Cas12a system at 37°C for 30 min produced a clearly visible fluorescence signal. Both methods demonstrated excellent specificity when tested against various bacterial strains. Furthermore, a one-step detection method combining RPA and CRISPR was developed, a mere 30-min reaction at 37°C is sufficient. In summary, the method combining RPA amplification with CRISPR/Cas12a detection offers better compatibility, and the one-step detection method effectively reduces the risk of aerosol contamination. It is well suited for rapid detection.