Mao, KangZhang, HuaRan, FangCao, HaoruiFeng, RidaDu, WeiLi, XiqingYang, Zhugen2023-10-252023-10-252023-10-17Mao K, Zhang H, Ran F, et al., (2024) Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater, Journal of Hazardous Materials, Volume 462, January 2024, Article Number 1327930304-3894https://doi.org/10.1016/j.jhazmat.2023.132793https://dspace.lib.cranfield.ac.uk/handle/1826/20442Wastewater 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.enAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/Antibiotic resistance geneCRISPR/Cas12aLoop-mediated isothermal amplificationBiosensorWastewaterArticle1873-3336