Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater

Abstract

Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 103 copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.