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Title: DEVELOPMENT OF A REAL-TIME PCR ASSAY FOR DETECTION OF SULFATE-REDUCING BACTERIA IN STORED SWINE MANURE AND SWINE FECES

Author
item Cook, Kimberly - Kim
item Whitehead, Terence
item Cotta, Michael

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/27/2004
Publication Date: 5/27/2004
Citation: Cook, K.L., Whitehead, T.R., Cotta, M.A. 2004. Development of a real-time PCR assay for detection of sulfate-reducing bacteria in stored swine manure and swine feces [abstract]. American Society for Microbiology. Paper No. Q-093.

Interpretive Summary:

Technical Abstract: Odor produced as a consequence of incomplete digestion of swine manure by bacteria has led to increased public concern and heightened tension between livestock producers and their rural neighbors. Sulfate-reducing bacteria (SRB) are a group of organisms responsible for production of hydrogen sulfide, an extremely odorous by-product of anaerobic metabolism. However, the concentration and types of SRB present in swine manure have not been characterized. Real-time, quantitative PCR permits rapid, sensitive detection of target organisms using fluorescently labeled probes. To better understand the occurrence of SRB in swine manure from underground pits and swine feces, an assay was developed to target the dissimilatory sulfite reductase (dsr) genes of two different groups of SRB. 16S rRNA primers specific for SRB, primers targeted to the dsrAB genes, and SRB enrichment cultures were used to determine the types of SRB present. Desulfovibrio sp. were identified from 16S rRNA clonal libraries from manure and from SRB enrichment cultures of manure and feces. Cloned dsrAB sequences from isolates from SRB enrichment cultures were similar to Desulfovibrio sp. (89%-91%). However, dsrAB clonal libraries from manure samples were dominated by clones with dsrAB sequences similar to either Desulfobulbus sp. (80%) or Desulfovibrio sp. (80%). A fluorescent TaqMan probe was developed to target both of these groups, but primers were designed to target either Group 1 (Desulfobulbus-like) or Group 2 (Desulfovibrio-like). A 161 bp fragment of the Group 1 or a 129 bp fragment of the Group 2 dsrA gene was targeted. Strong fluorescence was obtained, and linearity was observed over 6 orders of magnitude (10**2 to 10**8 copies of dsrA gene standard in PCR reaction) for both the Group 1 and the Group 2 assays. Based on analysis of manure samples, the number of copies of the dsrA gene may be as high as 7 X 10**7 copies per mL of sample. Results suggest that this real-time PCR assay represents a sensitive, specific new method for detection of SRB that will provide valuable, new information about the concentration of SRB in environmental samples.