A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters.

摘要: This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 dPCR (referred as hereon) uses same primer probe sequences its published individual quantitative (qPCR) counterparts. Likewise, water filtration DNA extraction procedures performed prior to qPCR are followed running dPCR. However, has several advantages over assays. Most important, eliminates need standard curve hence, associated bias variability, by direct targets. In addition, while duplexing (i.e. quantification) in often leads severe underestimation less abundant target sample, provides consistent both targets, whether quantified individually or simultaneously reaction. is also able tolerate inhibitor concentrations that one two orders magnitude higher than those tolerated qPCR. These make particularly attractive because it accurate repeatable information on general human-associated fecal contamination environmental waters without run separate Despite qPCR, upper limit with currently available instrumentation approximately four lower achievable Consequently, dilution needed measurement high organisms such typically observed following sewage spills.