Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate-reducing bacteria in stored swine manure

• Published in: Journal of applied microbiology Vol. 105; no. 6; pp. 2143 - 2152
• Main Authors: Spence, C, Whitehead, T.R, Cotta, M.A
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.12.2008; Blackwell Publishing Ltd; Blackwell
• Subjects: Animals; Biological and medical sciences; Desulfobulbus; Desulfovibrio; DNA Primers - genetics; DNA, Bacterial - analysis; dsrA; Fundamental and applied biological sciences. Psychology; Manure - microbiology; Microbiology; pig manure; polymerase chain reaction; Polymerase Chain Reaction - methods; quantitative analysis; real‐time PCR; RNA, Ribosomal, 16S - analysis; Sequence Analysis, DNA; sulfate-reducing bacteria; Sulfur-Reducing Bacteria - genetics; Sulfur-Reducing Bacteria - isolation & purification; Swine; swine manure; SYBR Green; Sulfate-reducing bacteria; Enumeration; Applied microbiology; SYBR Green; real-time PCR; Real time; Enumeration(counting); Pig; Polymerase chain reaction; dsrA; Vertebrata; Mammalia; Analysis method; Swine; Artiodactyla; Detection; Ungulata; Comparative study; swine manure; Quantitative analysis
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2008.03900.x

To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real-time PCR detection of sulfate-reducing bacteria (SRB) in stored swine manure. Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR-R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus-like Group 1 SRB in manure. The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.