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

被引:21
作者
Mao, Kang [1 ]
Zhang, Hua [1 ]
Ran, Fang [1 ]
Cao, Haorui [1 ]
Feng, Rida [1 ]
Du, Wei [2 ]
Li, Xiqing [3 ]
Yang, Zhugen [4 ]
机构
[1] Chinese Acad Sci, Inst Geochem, State Key Lab Environm Geochem, Guiyang 550081, Peoples R China
[2] Kunming Univ Sci & Technol, Fac Environm Sci & Engn, Kunming 650500, Peoples R China
[3] Peking Univ, Coll Urban & Environm Sci, Lab Earth Surface Proc, Beijing 100871, Peoples R China
[4] Cranfield Univ, Sch Water Energy & Environm, Cranfield MK43 0AL, England
基金
中国国家自然科学基金;
关键词
Antibiotic resistance gene; CRISPR/Cas12a; Loop-mediated isothermal amplification; Biosensor; Wastewater; NUCLEIC-ACID DETECTION; DIAGNOSTICS; SARS-COV-2;
D O I
10.1016/j.jhazmat.2023.132793
中图分类号
X [环境科学、安全科学];
学科分类号
08 ; 0830 ;
摘要
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 x 103 copies/mu L 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
引用
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页数:10
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