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Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

Hill VR, Narayanan J, Gallen RR, Ferdinand KL, Cromeans T, Vinjé J - Pathogens (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Division of Foodborne, Waterborne, and Environmental Diseases, 1600 Clifton Road NE, Mailstop D-66, Atlanta, GA 30329, USA. vhill@cdc.gov.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Flow diagram of optimized UNEX buffer protocol for the molecular detection of enteric microbes in water samples.
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pathogens-04-00335-f001: Flow diagram of optimized UNEX buffer protocol for the molecular detection of enteric microbes in water samples.

Mentions: The results of the technique evaluation experiments led to the development of the nucleic acid extraction, purification and analysis method identified in Figure 1.


Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

Hill VR, Narayanan J, Gallen RR, Ferdinand KL, Cromeans T, Vinjé J - Pathogens (2015)

Flow diagram of optimized UNEX buffer protocol for the molecular detection of enteric microbes in water samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493477&req=5

pathogens-04-00335-f001: Flow diagram of optimized UNEX buffer protocol for the molecular detection of enteric microbes in water samples.
Mentions: The results of the technique evaluation experiments led to the development of the nucleic acid extraction, purification and analysis method identified in Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Division of Foodborne, Waterborne, and Environmental Diseases, 1600 Clifton Road NE, Mailstop D-66, Atlanta, GA 30329, USA. vhill@cdc.gov.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus