Next generation sequencing reveals limitation of qPCR methods in quantifying emerging antibiotic resistance genes...

Occurrence of emerging antibiotic resistance genes (ARGs) in the environment, especially those conferring resistance to the last resort antibiotic carbapenems (bla_KPC) and colistin (mcr-1), has become an important environmental issue. Real-time polymerase chain reaction (qPCR) methods were commonly used to quantify emerging ARGs in the environment, with some studies reporting high abundance. Due to the high diversity of DNA templates and complexity in environmental samples, overestimation or even false-positive detection of target genes may occur due to potential non-specific amplification. This study compared the performance of dye-based qPCR and probe-based qPCR assays for the detection of bla_KPC and mcr-1 in activated sludge (AS) samples, which showed that the detection of bla_KPC and mcr-1 by the dye-based qPCR assays was likely false-positive when compared with probe-based qPCR results. Next generation sequencing (NGS) of the qPCR reactions identified primer dimers and non-specific amplicons as the primary causes for the false-positive detection. NGS also detected target amplicons in the negative reactions, indicating potential false-negative detection by the probe-based qPCR assays. Testing of spiked samples showed false-positive detection and overestimation by the dye-based qPCR assays primarily occurred at low concentrations of target DNA, while false-negative detection by probe-based qPCR was caused primarily by reduced amplification efficiencies in the environmental samples. Together, the results identified the limitations of the qPCR methods in complex environmental samples and demonstrated the remedial utility of NGS in quantifying emerging ARGs by qPCR. KEY POINTS: • Dye-based qPCR assays are prone to false-positive and overestimation at low substrate concentration. • Probe-based qPCR assays can experience false-negative detection due to environmental interference. • The combination of qPCR with NGS provides improved sensitivity and reliability in detecting low level ARGs in complex environmental matrices.