title: Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil.
abstract: In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus, and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the approximately 1 x 10(9) to approximately 1 x 10(4) colony forming units (CFU)/g soil range. The detection limit was improved to approximately 1 x 10(2) CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilisstrains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 x 10(2) CFU/g detection limit by days 28-105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.
authors: Department of Biology, Carleton University, Ottawa, ON K1S 5B6, Canada.
authors: Miguel A
authors: MA
authors: Providenti
authors: Melissa
authors: M
authors: Begin
authors: Samielle
authors: S
authors: Hynes
authors: Christine
authors: C
authors: Lamarche
authors: David
authors: D
authors: Chitty
authors: Jessica
authors: J
authors: Hahn
authors: Lee A
authors: LA
authors: Beaudette
authors: Rick
authors: R
authors: Scroggins
authors: Myron L
authors: ML
authors: Smith
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D001407:Bacillus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D017753:Ecosystem; D005819:Genetic Markers; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers