title: IMS-free DNA extraction for the PCR-based quantification of Cryptosporidium parvum and Giardia lamblia in surface and waste water.
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.
authors.affiliation: Environmental Engineering and Management, Asian Institute of Technology, Pathumthani, Thailand.
authors.forename: Alfredo J
authors.initials: AJ
authors.lastname: Anceno
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Katayama
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
authors.forename: Porntip
authors.initials: P
authors.lastname: Chavalitshewinkoon-Petmitr
authors.forename: Buyan
authors.initials: B
authors.lastname: Chuluun
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Shipin
mesh_terms: D000818:Animals; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D004247:DNA / Q000145:classification / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods*; D005838:Genotype; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D018189:Immunomagnetic Separation; D016133:Polymerase Chain Reaction / Q000379:methods; D014867:Water / Q000469:parasitology*
chemical_list: D014867:Water; D004247:DNA