Real Time PCR for the Rapid Detection of vanA Gene in Surface Waters and Aquatic Macrophyte by Molecular Beacon Probe

Abstract

Enterococci serve as an "indicator" of fecal contamination for recreational water quality. The vancomycin-resistant-enterococci (VRE) are emerging environmental contaminants in the surface waters. The aim ofthis study wasto develop a rapid and specific molecular beacon probe (MBP)-based real-time PCR assay for detection of vanA gene in surface waters and aquatic macrophyte. The limit of detection (LOD) of the MBP assay was 1 CFU/mL of VRE [r = 0.943; PCR efficiency = 99.7%] in 2-fold dilution format within 2.5 h and demonstrated high specificityfor environmental enterococci isolates exhibiting VanA phenotype (n=25). VRE were detected from downstream surface waters of the rivers impacted by point sources of pollution and recreational activities.The probe detected vanA gene in rootmat associated microbiota of E. crassipes (Mart) Solms. an aquatic nuisance weed, at eutrophic sites of the surface waters (ANOVA p < 0.001). In addition, the assay enabled detection of otherwise nondetectable vanA gene concentration in the upstream sites of two Indian rivers (Student's ttest p < 0.001). The MBP assay developed can be used for sensitive and rapid detection of VRE in surface waters and identification of nonpoint sources of pollution for implementation of preventive measures to protect human health.