Development of a method for rapid and simultaneous detection of antibiotic resistance genes in wastewater using a portable quantitative PCR device

Antimicrobial resistance has become a significant threat to public health, and wastewater treatment plants (WWTPs) serve as key reservoirs of antibiotic resistance genes (ARGs). Conventional quantitative PCR (qPCR) is widely used for the detection of ARGs; however, it poses the drawbacks of requiring specialized equipment and being relatively time-consuming. In this study, a method for the rapid and simultaneous detection of three ARGs (mecA, ampC, and mcr-1) in wastewater using the portable qPCR device, PicoGene® PCR1100, was developed. Fewer than 6.4 copies/reaction of the three ARGs were detected within 20 min using singleplex qPCR with PicoGene® PCR1100, with the limit of quantification and limit of detection comparable to those obtained by both the conventional qPCR system and the triplex qPCR assay on PicoGene® PCR1100. Moreover, a triplex qPCR workflow was optimized for the detection of the three target ARGs in wastewater samples, with the optimization including concentration and DNA extraction methods. Consequently, the polyethylene glycol precipitation method and the QIAamp DNA Mini Kit were determined as components of the optimal workflow. The optimal workflow was applied to wastewater monitoring, which revealed that the three target ARGs were successfully detected in 95 % (18/19) to 100 % (19/19) of the tested wastewater samples, with concentrations of between 4.6 ± 0.5 and 6.5 ± 0.2 log₁₀ copies/L. This study demonstrates the utility of triplex qPCR using PicoGene® PCR1100 as a valuable tool for environmental surveillance as it allows rapid and simultaneous detection of ARGs in wastewater, giving insight into their trends.