title: Bioinformatics and molecular biology for the quantification of closely related bacteria.
abstract: Molecular biological methods for mixed culture analysis outshine conventional culture-based techniques in terms of better sensitivity and reliability. The majority of these methods exploit the 16S rRNA sequences of the community DNA, which often fall short for the analysis of closely related microorganisms. This research details the development and validation of a comprehensive methodology to differentiate and quantitatively characterize two Pseudomonas species in a mixed culture. A bioinformatics tool based on whole-genome polymorphism comparison was used to identify marker sequences to differentiate the two bacteria using quantitative real-time PCR. The quantification of the two species was achieved through a correlation of the genomic DNA versus cell number (genomic DNA purification) and threshold cycle number versus genomic DNA (real-time PCR). Several factors including the limitation of genomic DNA purification, effects of substrate concentrations and growth phase on cellular DNA, and choice of simplex or duplex reaction for real-time PCR were considered and evaluated. The developed method was experimentally validated against synthetically constructed consortia.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore.
authors.forename: Karthiga
authors.initials: K
authors.lastname: Nagarajan
authors.forename: Kai-Chee
authors.initials: KC
authors.lastname: Loh
authors.forename: Sanjay
authors.initials: S
authors.lastname: Swarup
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D019295:Computational Biology / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D008967:Molecular Biology; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D012336:RNA, Ribosomal, 16S