AWWA WQTC69414

This powerpoint presentation begins by providing a brief overview of molecular testing of drinking water, and barriers to PCR use by drinking water utilities. AwwaRF Project 3108, "Improving Sample Preparation Methods for Molecular Techniques for Drinking Water Applications", is presented, having the following goals: to evaluate diverse nucleic acid extraction and purification techniques to develop an effective sample preparation method; demonstrate applicability to wide range of water samples and multiple types of organisms; determine ease, speed, and cost of method; and, perform multiple lab comparisons. The project's scope of work was to: perform a literature review and utility survey; obtain raw and finished water from six participating utilities; evaluate sample prep techniques and reagents using nuclease-free water and utility water; and, develop sample prep protocol and test via inter-laboratory validation study. Techniques evaluated included: Lysis buffer - chaotropic salts (e.g., guanidinium isothiocyanate), reducing agents (e.g., 2-mercaptoethanol, DTT, DTE), surfactants (anionic, nonionic, cationic, zwitterionic), and Lytic enzymes (e.g., proteinase K, lysozyme); physical disruption - bead beating, sonication; nucleic acid separation - silica column, microconcentrators, magnetic silica beads; PCR inhibition reduction - PVP, PVPP, Sephadex, Sepharose, Chelex-100; and, PCR facilitators. Presentation conclusions indicate the following: a universal sample prep method developed for molecular detection of viruses, vegetative bacteria, bacterial spores, and parasite (oo)cysts using PCR or RT-PCR; Lysis buffer developed for use with bead beating, followed by silica column and PVPP column processing; validation study demonstrated that method is readily and effectively implemented by utilities; no silver bullet for inhibitor removal; and, cost per 500-µL sample: ~$6.50. Includes tables, figures.