Advancing biosensing techniques for detection of antimicrobial resistance genes and antibiotics in water

Antimicrobial resistance (AMR) poses a significant global public health threat requiring urgent attention for surveillance of antibiotic resistance genes (ARGs) and antibiotics in the environment. In this PhD study, a real-time fluorescent detection assay of antimicrobial resistance genes (ARGs) was developed to specifically target two key ARGs, tet(M) and tet(x3) to detect tetracycline and tigecycline resistance respectively in water samples. Additionally, crassphage gene was investigated for anthropogenic activities since they also play a vital role in the AMR transmission. The advanced ARG detection assay was based on multiplexing recombinase polymerase amplification (RPA) and subsequent sequence-specific recognition by the trans-cleavage activity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a. The assay achieved limits of detection (LODs) of 1 copy µL⁻¹ for all three gene targets with an accuracy of 100% in spiked tap and surface water samples. Expanding the assay by including two additional end-point detection modalities, lateral flow assay (LFA) and voltametric detection, further demonstrate its versatility. LODs of 1 copy µL⁻¹ for tet(x3) and crassphage, and 10 copies µL⁻¹ for tet(M) (LFA) and 10 copies µL⁻¹ for all three targets (electrochemical) were reached. Validation against gold standard quantitative polymerase chain reaction (qPCR) using real water samples, including wastewater and drinking water samples, revealed a remarkable 100% accuracy rate. Antibiotics detection assay was conceptualised based on the amplification of hybridisation chain reaction (HCR) followed by CRISPR/Cas mediated cleavage within a DNA hydrogel matrix. This approach aimed to release electroactive methylene blue (MB) particles, detectable and quantifiable by square wave voltammetry (SWV). While unforeseen circumstances impeded full assay development, the preliminary data illustrated the viability of the proposed method, suggesting avenues for further research to develop rapid and onsite antibiotics detection methods.