Alfredo J. AncenoHiroyuki KatayamaEric R. HouptPorntip Chavalitshewinkoon-PetmitrBuyan ChuluunOleg V. ShipinAsian Institute of Technology ThailandUniversity of TokyoUniversity of VirginiaMahidol University2018-08-242018-08-242007-08-01International Journal of Environmental Health Research. Vol.17, No.4 (2007), 297-31013691619096031232-s2.0-34447130252https://repository.li.mahidol.ac.th/handle/20.500.14594/24459Extremely limited knowledge exists on the occurrence of protozoan pathogens in surface and waste water in the developing world. The article addresses one of the major reasons for this: prohibitively costly immunomagnetic separation (IMS) and commercial DNA extraction kits are required for the pathogen detection. As the presence of inhibitory substances critically impedes the polymerase chain reaction (PCR)-based detection of Cryptosporidium and Giardia in environmental samples, several direct DNA extraction methods based on the combination of physico-chemical means were evaluated in terms of reducing the impact of PCR inhibitors present in (oo)cyst-spiked water concentrates. Modifications that included the use of guanidine thiocyanate as a lysis agent and a sonication step were found to be more efficient in extracting DNA from (oo)cysts, while treatment with Chelex 100 chelating resin at post-lysis proved to be effective in the removal of the PCR inhibitors rather than the inclusion of the PCR facilitators during thermocycling. Direct DNA extraction protocol at a substantially reduced cost is proposed for the use in the PCR-based detection/quantification of the pathogens. © 2007 Taylor & Francis.Mahidol UniversityEnvironmental ScienceMedicineArticleSCOPUS10.1080/09603120701372573