title: Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification.
abstract: The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R²=0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.
authors.affiliation: LUNAM Université, Oniris, UMR 1300 Biologie, épidémiologie et analyse des risques, Nantes, F-44307, France. leblanc.maridor@gmail.com
authors.forename: Mily
authors.initials: M
authors.lastname: Leblanc-Maridor
authors.forename: Amélie
authors.initials: A
authors.lastname: Garénaux
authors.forename: François
authors.initials: F
authors.lastname: Beaudeau
authors.forename: Bérangère
authors.initials: B
authors.lastname: Chidaine
authors.forename: Henri
authors.initials: H
authors.lastname: Seegers
authors.forename: Martine
authors.initials: M
authors.lastname: Denis
authors.forename: Catherine
authors.initials: C
authors.lastname: Belloc
mesh_terms: D000818:Animals; D058491:Bacterial Load / Q000379:methods* / Q000592:standards; D002167:Campylobacter / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013552:Swine; D044124:Yersinia ruckeri / Q000235:genetics
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes