Abstract |
Extremely 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.
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Authors | Alfredo J Anceno, Hiroyuki Katayama, Eric R Houpt, Porntip Chavalitshewinkoon-Petmitr, Buyan Chuluun, Oleg V Shipin |
Journal | International journal of environmental health research
(Int J Environ Health Res)
Vol. 17
Issue 4
Pg. 297-310
(Aug 2007)
ISSN: 0960-3123 [Print] England |
PMID | 17613093
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
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Topics |
- Animals
- Cryptosporidium parvum
(genetics, isolation & purification)
- DNA
(classification, isolation & purification)
- Environmental Monitoring
(methods)
- Genotype
- Giardia lamblia
(genetics, isolation & purification)
- Immunomagnetic Separation
- Polymerase Chain Reaction
(methods)
- Water
(parasitology)
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