Protocol

tDNA-PCR Followed by Automated Fluorescent Capillary Electrophoresis for Identification of Bacterial Species

¹ Laboratory for Veterinary Microbiology, Department of Pathology, Bacteriology and Poultry Diseases, Ghent University, 9000 Gent, Belgium
² Nano Engineered Component Science (NEXT-NS), IMEC vzw, 3000 Leuven, Belgium
³ Laboratory Bacteriology Research (LBR), Department of Clinical Chemistry, Immunology, Microbiology, Ghent University, 9000 Gent, Belgium

⁴Corresponding author (Mario.Vaneechoutte{at}UGent.be)

INTRODUCTION

Transfer RNA intergenic spacer length polymorphism analysis (tDNA-PCR) is a simple and reproducible polymerase chain reaction (PCR) technique for identification of bacteria at the species or even subspecies level. The primers used in the PCR are based on conserved sequences located at the edges of the tRNA genes. Because the selected consensus primers are directed outwardly, the intergenic spacers are amplified rather than the genes themselves. With each PCR, several amplicons of different lengths are obtained, because several intergenic spacers are present in each bacterial genome. The patterns thus obtained are identical within species, but differ between distinct species, and as a result, can be used for identification of bacterial species. The amplicons are separated using high-resolution (1 bp) electrophoresis (e.g., fluorescent capillary electrophoresis) and immediately digitized as tables composed of numerical lengths (expressed in base pairs) and peak intensities. For identification, the resulting peak pattern can be compared with a large database of patterns of well-identified bacterial strains, using an in-house-developed software package that is available online. New patterns (linked to the correct species name, which can be obtained, e.g., after 16S rRNA gene sequence determination) can be added to expand the database further. This protocol describes tDNA-PCR, followed by automated fluorescent capillary electrophoresis to identify bacterial species.