Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production

Abstract

The microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in groundwater samples. This study focuses on the optimization, validation and application of a qPCR method combined with propidium monoazide (PMA) treatment to exclude DNA from dead cells. A first screening consisting of six primer sets targeting single and multi-copy of E. coli were tested to evaluate the sensitivity of the assay. After that, four primer sets were selected, combined with PMA treatment and their capacity to distinguish viable cells when combined with a background of dead cells was assessed. A primer set targeting the 23S rRNA gene was 10-fold more sensitive than the rest of primers, enabling the detection of low concentrations of viable E. coli cells. This assay also exhibited good repeatability and reproducibility, which indicates the robustness of the method. Optimized and validated PMA-qPCR was used to enumerate E. coli in environmental samples including irrigation water and fresh produce. The results were compared with the levels quantified using qPCR and cultivation-based techniques. Counts of E. coli using plate count assay were significantly lower than the levels obtained by molecular techniques (PMA-qPCR and qPCR) in both irrigation water and fresh produce. E. coli PMA-qPCR enumeration method showed similar results as qPCR quantification, although the PMA-qPCR treatment seemed to a good alternative to distinguish between viable and dead cells. It can be concluded that the optimized PMA-qPCR assay can be used by the industry in microbial sampling programs, helping them with the implementation of Good Agricultural Practices (GAP).