Details:

Alternative Title:
Pathogens
Personal Author:
Hill, Vincent R. ; Narayanan, Jothikumar ; Gallen, Rachel R. ; Ferdinand, Karen L. ; Cromeans, Theresa ; ... More +
Hill, Vincent R. ; Narayanan, Jothikumar ; Gallen, Rachel R. ; Ferdinand, Karen L. ; Cromeans, Theresa ; Vinjé, Jan Less -
Description:
Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.
Subjects:
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Article DNA Drinking Water Extraction Nucleic Acid PCR Facilitators Purification RNA Water Testing
Source:
Pathogens. 2015; 4(2):335-354.
Pubmed ID:
26016775
Pubmed Central ID:
PMC4493477
Document Type:
Journal Article
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:6a0c0c7a8ccbb9a25bb07cd4ccb32f7a1c03c409135218c304283a48f050cf3a
Download URL:
https://stacks.cdc.gov/view/cdc/31979/cdc_31979_DS1.pdf
File Type:

Supporting Files

• 	license.txt	txt
  	pathogens-04-00335-g001.gif	gif
  	pathogens-04-00335-g001.jpg	jpeg
  	pathogens-04-00335.nxml	txt

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