Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

Pathogens

Vincent R. Hill¹  Rachel R. Gallen¹  Jothikumar Narayanan¹  Theresa Cromeans²  Karen L. Ferdinand²  Jan Vinjé² 

[1] 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;Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Viral Diseases, Atlanta, GA 30329, USA;

关键词: drinking water;    water testing;    nucleic acid;    DNA;    RNA;    extraction;    purification;    PCR facilitators;   

DOI  :  10.3390/pathogens4020335

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown