Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.
Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.
Lemarchand
K
K
National Research Council of Canada, Biotechnology Research Institute, Montréal, QC, Canada. Karine_Lemarchand@uqar.qc.ca
Berthiaume
F
F
Maynard
C
C
Harel
J
J
Payment
P
P
Bayardelle
P
P
Masson
L
L
Brousseau
R
R
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014865:Waste Disposal, Fluid / Q000379:methods*; D014871:Water Microbiology
D004269:DNA, Bacterial