Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline\ud resistance genes in agricultural ecosystems

摘要

Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora\udin pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we\uddeveloped and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin\ud(strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in\udplant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly\udfrom bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry\udtreatment. The genes most frequently identified in soils pre- and post-slurry treatment were strB, aadA, tetB\udand tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative\udconcentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry\udtreatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification\udof antibiotic resistance genes in complex, environmental sample matrices.