Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region.
OBJECTIVE
Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples.


RESULTS
Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples.


CONCLUSIONS
The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples.


CONCLUSIONS
Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.
National Water Research Institute, Aquatic Ecosystem Protection Research Division, Environment Canada, Burlington, Ontario, Canada.
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Khan
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D001483:Base Sequence; D002167:Campylobacter / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017000:Campylobacter coli / Q000235:genetics; D016123:Campylobacter jejuni / Q000235:genetics; D044863:Campylobacter lari / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D004783:Environmental Microbiology; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012338:RNA, Ribosomal, 23S / Q000032:analysis; D012680:Sensitivity and Specificity
D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S