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CHARACTERIZATION OF VIRAL RNA EXTRACTION EFFICIENCY FROM ENVIRONMENTAL WATERS
Citation:
OLSZEWSKI, J., N. ADCOCK, A. YU, K. KELTY, I. KATZ, D. RUSSELL, A. LINCOFF, R. GIGGER, S. SPENCE, AND S. HARRIS. CHARACTERIZATION OF VIRAL RNA EXTRACTION EFFICIENCY FROM ENVIRONMENTAL WATERS. Presented at AWWA's Water Quality and Technology Conference, Denver, CO, November 05 - 09, 2006.
Description:
Inhibition of PCR by environmental factors is a common problem affecting the sensitive detection of pathogenic microorganisms in environmental waters. This inhibition is caused by one of three mechanisms: 1) failure to lyse the microorganism, 2) degradation or sequestering of the nucleic acid following lysis, or 3) inhibition of DNA polymerase during amplification. One solution to overcome these problems is the use of commercially available kits designed for highly efficient extraction and purification of nucleic acids. These kits are designed to overcome these three inhibitory mechanisms by using: (1) optimized chemicals and procedures to ensure maximum lysis of microorganisms, (2) concentration columns that bind released nucleic acids and allow them to be washed to remove inhibitory substances, or (3) a combination of these two approaches. However, while several commercial kits are available for the extraction of viral nucleic acids, none are designed or optimized for use in environmental water samples. Previous research has also shown that extraction efficiency in different water sources can be quite variable and effect detection efficiency by molecular techniques, such as RT-PCR. In order to characterize the extraction efficiency of five commercial RNA extraction kits, environmental water samples (drinking, surface, ground, sea and sewage) from six geographically diverse regions of the U.S. were obtained and spiked with a known concentration of poliovirus 2, reovirus 1 and bacteriophage MS2. Water samples were taken and extracted using each RNA extraction kit and analyzed by real-time RT-PCR to determine extraction efficiency. In subsequent experiments, ten liter water samples were concentrated using a hollow-fiber ultrafiltration system and spiked with poliovirus 2, reovirus 1 and bacteriophage MS2. As with initial testing, water samples were taken, extracted using each kit and analyzed by real-time RT-PCR. The objective of this research is to characterize the extraction efficiency of various commercial viral RNA extraction kits and their ability to produce high quality nucleic acids suitable for molecular techniques and under various water conditions.