AWWA WQTC64115

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: failure to lyse the microorganism; degradation or sequestering of the nucleic acid following lysis; or, 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: optimized chemicals and procedures to ensure maximum lysis of microorganisms; concentration columns that bind released nucleic acids and allow them to be washed to remove inhibitory substances; or, a combination of these two approaches. 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 RTPCR. In order to characterize the extraction efficiency of four commercial RNA extraction kits, 2 L environmental water samples (drinking, surface, ground, marine and sewage) from six geographically diverse regions of the U.S. were obtained and spiked with a ~1000 pfu/ml concentration of poliovirus 2 and bacteriophage MS2. Samples were incubated and mixed thoroughly at room temperature to ensure complete mixing of the virus. A 15 ml aliquot was taken from each water sample for analysis by real-time RT-PCR and cell culture. Water sample aliquots were then extracted using each RNA extraction kit and analyzed by real-time RT-PCR. In addition, a distilled water sample spiked with ~1000 pfu/ml of virus, extracted and analyzed by real-time RT-PCR. For determining extraction efficiency, the extracted distilled water sample was considered to be 100%. For the RTPCR reaction, 5 µl of sample were amplified using ABI TaqMan one-step RT-PCR mastermix using primers and probes. Extractions were also examined by a real-time PCR inhibition assay to determine if the extractions contained inhibitors. The inhibition assay used 5 µl of sample added to a TaqMan one-step mastermix containing a control DNA sample, primers and probes provided in the ABI exogenous internal control kit. Five positive control samples containing the inhibitor contained in the kit and five negative controls containing just water were also included. All real-time assay were run on ABI 96-well optical plates. Includes tables.