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This powerpoint presentation begins by providing a brief overview of molecular testing of drinkingwater, and barriers to PCR use by drinkingwater utilities. AwwaRF Project 3108, "Improving Sample Preparation Methods forMolecular Techniques for Drinking WaterApplications", is presented, having the following goals: to evaluate diverse nucleic acid extraction andpurification techniques to develop an effectivesample preparation method;demonstrate applicability to wide range of watersamples 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 participatingutilities;evaluate sample prep techniques and reagents usingnuclease-free water and utility water; and,develop sample prep protocol and test via inter-laboratoryvalidation 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), andLytic 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 developedfor molecular detection of viruses, vegetativebacteria, bacterial spores, and parasite(oo)cysts using PCR or RT-PCR;Lysis buffer developed for use with beadbeating, followed by silica column and PVPPcolumn processing;validation study demonstrated that method isreadily and effectively implemented by utilities;no silver bullet for inhibitor removal; and,cost per 500-L sample: ~$6.50. Includes tables, figures.

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Edition: Vol. - No. Published: 11/01/2008 Number of Pages: 27File Size: 1 file , 1 MB