Validation of hollow fiber ultrafiltration and real-time PCR using bacteriophage PP7 as surrogate for the quantification of viruses from water samples

A quantitative real-time TaqMan^® PCR system for Pseudomonas aeruginosa bacteriophage PP7 was designed to detect PP7 as surrogate in performance tests of 2 hollow fiber ultrafiltration systems in series. Fifty-six storm water samples from 21 sites representing agricultural, urban and highway locations in California were collected. The optimized procedure gave recoveries of spiked PP7 of 64±4.8% (mean±SEM). The PP7 assay was validated over 5 orders of magnitude with an assay limit of detection of 5 gene copies per reaction volume. Sample-dependent variables like enzymatic inhibition during PCR analysis, filtration recovery and extraction efficiency were quantified and incorporated to calculate a specific sample limit of detection (S_LOD) for the spiked surrogate PP7. S_LOD values were highly variable among samples; they were independent of physicochemical parameters including conductivity, turbidity, total suspended solids and pH but strongly correlated with the dilution factor required to relieve enzymatic inhibition during PCR analysis. To determine actual gene copies of PP7, a dilution approach was developed that involves assaying several dilutions within a range where inhibitors do not affect the efficiency of amplification and linear regression to determine the theoretical C_t value when there is no inhibition. For the detection of viral pathogens, an internal standard like PP7 can be used to calculate filtration recoveries when quantifying pathogens and to determine whether filtration or inhibitor concentration affect nucleic acid extraction efficiency. Additionally, by defining S_LOD values per sample and pathogenic organism analyzed, it should be possible to critically investigate the absence of detects for a particular pathogen and determine probabilities of risk associated with a specific sample limit of detection.