IMS-free DNA extraction for the PCR-based quantification of Cryptosporidium parvum and Giardia lamblia in surface and waste water.
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.
Anceno
Alfredo J
AJ
Environmental Engineering and Management, Asian Institute of Technology, Pathumthani, Thailand.
Katayama
Hiroyuki
H
Houpt
Eric R
ER
Chavalitshewinkoon-Petmitr
Porntip
P
Chuluun
Buyan
B
Shipin
Oleg V
OV
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*
D014867:Water; D004247:DNA