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Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target.
- Source :
-
Journal of microbiological methods [J Microbiol Methods] 2006 Jan; Vol. 64 (1), pp. 46-62. Date of Electronic Publication: 2005 Aug 19. - Publication Year :
- 2006
-
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.
- Subjects :
- Animals
Chickens microbiology
Clostridium perfringens classification
Clostridium perfringens genetics
Colony Count, Microbial methods
DNA Primers genetics
Enterococcus isolation & purification
Escherichia coli isolation & purification
Gastrointestinal Contents chemistry
Genome, Bacterial
Lactobacillus isolation & purification
Lactobacillus acidophilus isolation & purification
Polymerase Chain Reaction methods
Reference Standards
Reproducibility of Results
Statistics as Topic
Swine microbiology
Chaperonin 60 genetics
Clostridium perfringens growth & development
Colony Count, Microbial veterinary
Gastrointestinal Contents microbiology
Gastrointestinal Tract microbiology
Subjects
Details
- Language :
- English
- ISSN :
- 0167-7012
- Volume :
- 64
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of microbiological methods
- Publication Type :
- Academic Journal
- Accession number :
- 16112762
- Full Text :
- https://doi.org/10.1016/j.mimet.2005.04.006