Gene Amplification and Sequencing for Bacterial Identification

Abstract

Abstract The most important task of the clinical microbiology laboratory is to accurately identify the responsible pathogens to species level. Phenotypic methods for bacterial identification are associated with many problems, such as strains with unusual biochemical profiles and slowly growing bacterial species. In the past few decades, gene sequencing has evolved to be an objective method for bacterial identification. Among the various gene targets, the 16S rRNA gene is the most common target used. However, for some groups of bacteria, 16S rRNA gene sequencing is not discriminative enough for discriminating closely related bacterial species. In such cases, other additional genes, such as groEL and rpoB, are used as the gene targets. In addition to identification, gene sequencing has led to the discovery of numerous novel bacterial genera and species. In the last few years, user-friendly databases and software have been developed for analysing 16S rRNA gene sequences, which has enabled the more common use of this technology in clinical microbiology laboratories feasible. Such databases have been validated using complete bacterial genome sequences. Moreover, the availability of abundant complete bacterial genome sequences has also facilitated the design of polymerase chain reaction primers for specific amplification of particular bacterial species for identification or directly from clinical or environmental specimens. Although matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry is an emerging technology for bacterial identification in clinical laboratories, it is not feasible to manually interpret the raw data in case of ambiguous results. Therefore, gene sequencing or a polyphasic approach for bacterial identification is still the standard to resolve difficult cases.