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Inhibition of PCR by environmental factors is a common problem affecting the sensitivedetection of pathogenic microorganisms in environmental waters. This inhibition iscaused by one of three mechanisms: failure to lyse the microorganism; degradationor sequestering of the nucleic acid following lysis; or, inhibition of DNA polymeraseduring amplification. One solution to overcome these problems is the use ofcommercially available kits designed for highly efficient extraction and purification ofnucleic acids. These kits are designed to overcome these three inhibitory mechanisms byusing: optimized chemicals and procedures to ensure maximum lysis ofmicroorganisms; concentration columns that bind released nucleic acids and allowthem to be washed to remove inhibitory substances; or, a combination of these twoapproaches. While several commercial kits are available for the extraction of viralnucleic acids, none are designed or optimized for use in environmental water samples.Previous research has also shown that extraction efficiency in different water sources canbe 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) fromsix geographically diverse regions of the U.S. were obtained and spiked with a ~1000pfu/ml concentration of poliovirus 2 and bacteriophage MS2. Samples were incubatedand mixed thoroughly at room temperature to ensure complete mixing of the virus. A 15ml aliquot was taken from each water sample for analysis by real-time RT-PCR and cellculture. Water sample aliquots were then extracted using each RNA extraction kit andanalyzed by real-time RT-PCR. In addition, a distilled water sample spiked with ~1000pfu/ml of virus, extracted and analyzed by real-time RT-PCR. For determining extractionefficiency, the extracted distilled water sample was considered to be 100%. For the RTPCRreaction, 5 l of sample were amplified using ABI TaqMan one-step RT-PCRmastermix using primers and probes. Extractions were alsoexamined by a real-time PCR inhibition assay to determine if the extractions containedinhibitors. The inhibition assay used 5 l of sample added to a TaqMan one-stepmastermix containing a control DNA sample, primers and probes provided in the ABIexogenous 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.

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Edition: Vol. - No. Published: 11/01/2006 Number of Pages: 3File Size: 1 file , 310 KB