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.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 1 MB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 27 |
| Published : | 11/01/2008 |
