A Heat Pretreatment Method to Improve the RT-qPCR Detectability of Plasmid-Borne Antibiotic Resistance Genes in Environmental Samples

The dissemination of antibiotic resistance genes (ARGs) has garnered significant attention. Our study revealed that nearly 90% of the supercoiled form plasmids in laboratory standard ARGs vector plasmids were undetectable by direct detection using Quantitative Real-time polymerase chain reaction (qPCR). We propose a pretreatment heating at 75 °C for 10 min to enhance the efficiency of qPCR detection for plasmid borne ARGs. Treatment of the pBR322 plasmid using this method enhanced the qPCR detectability of tetA (carried by pBR322 plasmid) from 14.1% to 69.1%. Subsequently, we extracted DNA from water samples collected from the Songhua River and compared the relative abundance of ARGs in the samples before and after the heat pretreatment. The results revealed that the relative abundance of approximately 56% of the ARGs increased, the majority of which are located on plasmids. This finding indicates that direct detection using qPCR could lead to an underestimation of the levels of contamination associated with plasmid-borne ARGs. In addition, we investigated that the change of vector plasmid structure in the ARGs degradation experiment can seriously interfere with the qPCR quantification of ARGs, thereby presenting a challenge to accurately assessing the degradation rate of ARGs.