Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay

To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeti...

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Bibliographic Details
Published in:Journal of applied microbiology 2010-07, Vol.109 (1), p.334-347
Main Authors: Weidhaas, J.L, Macbeth, T.W, Olsen, R.L, Sadowsky, M.J, Norat, D, Harwood, V.J
Format: Article
Language:English
Subjects:
Agricultural wastes
Animals
Aquatic birds
Bacteria
Biological and medical sciences
Brevibacterium
Brevibacterium - classification
Brevibacterium - genetics
Brevibacterium avium
Cattle - microbiology
Chickens - microbiology
Colony Count, Microbial
Deoxyribonucleic acid
DNA
DNA, Bacterial - genetics
Environmental Monitoring - methods
faecal contamination
Feces
Feces - microbiology
Fundamental and applied biological sciences. Psychology
Genes, Bacterial
Humans
Land pollution
Litter
Markers
microbial source tracking
Microbiology
Microorganisms
Nucleotide sequence
Nucleotides
Phylogeny
Polymerase chain reaction
Polymerase Chain Reaction - methods
Polymorphism, Restriction Fragment Length
Poultry
Poultry - microbiology
qPCR
RNA, Ribosomal, 16S - genetics
rRNA 16S
Sediment pollution
Sewage
Sewage - microbiology
Soil - analysis
Soil contamination
Soil Microbiology
Soil testing
Soils
Swine - microbiology
United States
waste disposal
water quality
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Summary:To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10⁷-10⁹ gene copies per gram in soiled litter, up to 10⁵ gene copies per gram in spread-site soils, and 10⁷ gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.
ISSN:1364-5072
1365-2672
DOI:10.1111/j.1365-2672.2010.04666.x