title: Evaluation of real-time PCR assays and standard curve optimisation for enhanced accuracy in quantification of Campylobacter environmental water isolates.
abstract: Campylobacter is a major public health and economic burden in developed and developing countries. This study evaluated published real-time PCR (qPCR) assays for detection of Campylobacter to enable selection of the best assays for quantification of C. spp. and C. jejuni in environmental water samples. A total of 9 assays were compared: three for thermotolerant C. spp. targeting the 16S rRNA and six for C. jejuni targeting different genes. These assays were tested in the wet-lab for specificity and sensitivity against a collection of 60, genetically diverse, Campylobacter isolates from environmental water. All three qPCR assays targeting C. spp. were positive when tested against the 60 isolates, whereas, assays targeting C. jejuni differed among each other in terms of specificity and sensitivity. Three C. jejuni-specific assays that demonstrated good specificity and sensitivity when tested in the wet-lab showed concordant results with in silico-predicted results obtained against a set of 211 C. jejuni and C. coli genome sequences. Two of the assays targeting C. spp. and C. jejuni were selected to compare DNA concentration estimation, using spectrophotometry and digital PCR (dPCR), in order to calibrate standard curves (SC) for greater accuracy of qPCR-based quantification. Average differences of 0.56±0.12 and 0.51±0.11 log fold copies were observed between the spectrophotometry-based SC preparation and the dPCR preparation for C. spp. and C. jejuni, respectively, demonstrating an over-estimation of Campylobacter concentration when spectrophotometry was used to calibrate the DNA SCs. Our work showed differences in quantification of aquatic environmental isolates of Campylobacter between qPCR assays and method-specific bias in SC preparation. This study provided an objective analysis of qPCR assays targeting Campylobacter in the literature and provides a framework for evaluating novel assays.
authors.affiliation: Public Health Agency of Canada, NML at Saint-Hyacinthe, Saint-Hyacinthe, QC J2S8E3, Canada; Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC J2S2M2, Canada.
authors.forename: Maxime
authors.initials: M
authors.lastname: Gosselin-Théberge
authors.affiliation: Public Health Agency of Canada, NML at Lethbridge, Lethbridge, AB T1J 3Z4, Canada.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Taboada
authors.affiliation: Public Health Agency of Canada, NML at Saint-Hyacinthe, Saint-Hyacinthe, QC J2S8E3, Canada; Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC J2S2M2, Canada. Electronic address: rebecca.guy@phac-aspc.gc.ca.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Guy
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods; D002138:Calibration; D002167:Campylobacter / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D002169:Campylobacter Infections / Q000382:microbiology*; D017000:Campylobacter coli / Q000235:genetics / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000235:genetics / Q000302:isolation & purification; D002645:Chickens / Q000382:microbiology; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000235:genetics; D005243:Feces / Q000382:microbiology; D011201:Poultry Diseases / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Assay comparison; Campylobacter; Digital PCR; Microbial in silico typing; Real-time PCR