title: Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target.
abstract: We used qPCR and the target gene chaperonin-60 (cpn60) to enumerate Clostridium perfringens genomes in DNA extracts from contents of the chicken gastrointestinal tract with the aim of optimizing this methodology to enumerate any bacterium of interest. To determine the most accurate protocols for determining target species abundance, we compared various DNA extraction methods in combination with four methods for producing standard curves. Factors affecting accuracy included the co-purification of PCR inhibitors and/or fluorescence quenchers and the yield of target DNA in the extract. Anion exchange chromatography of the spiked test samples enabled accurate enumeration of C. perfringens using a standard curve comprised of a plasmid containing a fragment of C. perfringens cpn60. We used qPCR to enumerate C. perfringens and other intestinal bacteria in ileum and cecum samples from chickens that had been challenged with C. perfringens and compared the results with viable counts on corresponding selective agars. We conclude that qPCR-based molecular enumeration of target species in the gastrointestinal tract is feasible, but care must be taken in order to mitigate the effects of confounding factors that can affect the apparent cell count.
authors.affiliation: Department of Animal and Poultry Science, University of Saskatchewan, 51 Campus Drive, Saskatoon SK, Canada, S7N 5A8.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Dumonceaux
authors.forename: Janet E
authors.initials: JE
authors.lastname: Hill
authors.forename: Seth A
authors.initials: SA
authors.lastname: Briggs
authors.forename: Kingsley K
authors.initials: KK
authors.lastname: Amoako
authors.forename: Sean M
authors.initials: SM
authors.lastname: Hemmingsen
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Van Kessel
mesh_terms: D000818:Animals; D018834:Chaperonin 60 / Q000235:genetics*; D002645:Chickens / Q000382:microbiology; D003016:Clostridium perfringens / Q000145:classification / Q000235:genetics / Q000254:growth & development*; D015169:Colony Count, Microbial / Q000379:methods / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D016983:Enterococcus / Q000302:isolation & purification; D004926:Escherichia coli / Q000302:isolation & purification; D005766:Gastrointestinal Contents / Q000737:chemistry / Q000382:microbiology*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D016680:Genome, Bacterial; D007778:Lactobacillus / Q000302:isolation & purification; D007779:Lactobacillus acidophilus / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012015:Reference Standards; D015203:Reproducibility of Results; D013223:Statistics as Topic; D013552:Swine / Q000382:microbiology
chemical_list: D018834:Chaperonin 60; D017931:DNA Primers