Waterborne pathogen detection by use of oligonucleotide-based microarrays.
A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.
Maynard
Christine
C
National Research Council of Canada, Biotechnology Research Institute, 6100 Ave. Royalmount, Montreal, Quebec, Canada H4P 2R2.
Berthiaume
Frédéric
F
Lemarchand
Karine
K
Harel
Josée
J
Payment
Pierre
P
Bayardelle
Paul
P
Masson
Luke
L
Brousseau
Roland
R
D018834:Chaperonin 60 / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D009838:Oligodeoxyribonucleotides; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics; D014871:Water Microbiology; D015008:Yersinia enterocolitica / Q000235:genetics
D018834:Chaperonin 60; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012336:RNA, Ribosomal, 16S