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Microbiological Detection Systems for Molecular Analysis of Environmental Water and Soil Samples
Citation:
KEELEY, ANN AND Q. Wu. Microbiological Detection Systems for Molecular Analysis of Environmental Water and Soil Samples. Presented at the Society of Industrial Microbiology and Biotechnology Annual Meeting, San Francisco, CA, August 01 - 05, 2010.
Impact/Purpose:
This work focused on the development, modification, and optimization of molecular tools in conjunction with pathogens seeded samples.
Description:
Multiple detection systems are being targeted to track various species and genotypes of pathogens found in environmental samples with the overreaching goal of developing analytical separation and detection techniques for Salmonella enterica Serovars Typhi, Cryptosporidium parvum, and Giardia lamblia. This work focused on the development, modification, and optimization of molecular tools in conjunction with pathogens seeded samples. Quantitative PCR-based assays were used due to their rapidity, preciseness, and higher sensitivity as compared to conventional PCR techniques. Since many Q-PCR published data have indicated varying degrees of specificities, we have designed, optimized, and tested primer/probe sets and their running conditions rather than relying on the available primers. EPA method 1623 was used for Immunomagnetic separation (IMS) of oocysts and cysts from water samples. DNA extraction from five different soils seeded with S. enterica Serovars Typhi, Cryptosporidium parvum, and Giardia lamblia were compared. The effect of bovine serum albumin (BSA) and polyvinylpyrrolidone (PVP) was tested to alleviate PCR inhibitors during Q-PCR. IAC was generated for analysis of samples spiked with S. enterica Serovars Typhi, and C. parvum in real time PCR. The results have shown that primers and probes were specific for S. enterica Serovars Typhi and a lower Ct value was observed in DNA extracted using InstaGene matrix for environmental waters seeded with S. enterica Serovars Typhi and DNA grade water spiked with C. parvum than other methods tested. On the other hand, PowerSoil DNA isolation kit was more effective in DNA extraction for soil samples seeded with S. enterica Serovars Typhi and C. parvum than other kits tested. A modification to PowerSoil indicated that the sample incubation at 70°C might help to lyse the cells. The minimum detection limit of S. enterica Serovars Typhi was 400 cells in water (0.5 ml) and soils (0.5 g) while C. parvum was observed in soil samples (0.5 g) seeded with 5 oocysts of C. parvum using real time PCR.
