Ribosomal ribonucleic acid sequence analysis and hybridization technology in the identification of microorganisms

Abstract

Conventional diagnostic methods for identifying microorganisms rely on differences in phenotypic properties. However these methods have often proved as unreliable and time-consuming. Our current knowledge on the phylogenetic relationship of bacteria is based upon comparative sequence analysis of 16S and 23S rRNA. These rRNA molecules contain regions of highly conserved and less conserved sequences. Oligonucleotides that are complementary to universally conserved regions close to the 5' and 3' termini of the 16S rRNA genes are used for the in vitro amplification of these genes. Small amounts of a cell suspension can be directly used for the in vitro amplification, without prior lysis of cells, and extraction of nucleic acids. Comparison with known 16S rRNA sequences identifies the organism on a phylogenetic basis. A comprehensive 16S rRNA data bank and a correct alignment of the sequences based on their primary and secondary structures are a prerequisite for a reliable identification of the organism. Complementary to more or less conserved sequence regions of 16S and 23S rRNAs, oligonucleotides (nucleic acid probes) can be designed with specificities ranging from species specificity to a universal probe that will hybridize with any cellular life form. A nucleic acid probe is a fragment of a nucleic acid that binds (hybridizes) to complementary DNA or RNA (target nucleic acid). The reactions are highly specific so that probes will bind to their complementary sequences, even if these sequences account for only a small fraction of the target nucleic acid. The nucleic acid probe technique consists of four steps. First, the probe has to be labelled with a radioactive or nonradioactive detector group (enzymes, haptens, fluorescent groups). Second, the target nucleic acid has to be extracted or the cells have to be made permeable to the probe. Third, the probe has to react with the target nucleic acid (hybridization) and the unbound or non-specifically-bound probe has to be removed (washing). Fourth, the specific hybrids are detected by measuring the amount of labelled probe bound to the target nucleic acid. Using rRNA as target nucleic acid also considerably increases the sensitivity of the test since rRNAs are present in high copy numbers (up to l0 s copies per cell) in growing bacterial cells. Probes directed against rRNA allow rapid classification of unknown isolates. As a first step, a universal probe is applied to provide a rough estimate of the amount of target nucleic acid present in the hybridization reaction. The hybrids between the universal probe and the target nucleic acid are denatured, the universal probe is removed and an eubacterial probe is applied. By repeating this procedure using increasingly specific probes it is possible to identify unknown isolates at the genus, or even species level, in just a few steps. Different formats can be used for probe assays. For the dotblot hybridization assay small amounts of target nucleic acids have to be extracted and bound to nylon, nitrocellulose or teflon membranes. Colony hybridization is a convenient, rapid, and simple method for analysing mixed cultures. The colonies are transferred to membranes and are lysed. Gram-negative bacteria can be easily lysed on filters using simple alkali treatment, whereas Gram-positive bacteria require an additional heating step. For the in situ identification of individual whole cells it is necessary to make the cells permeable to oligonucleotide probes reacting with rRNA. Currently, the method works predominantly with Gram-negative bacteria since the permeabilization of Gram-positive bacteria is more difficult. Fluorescentlabelled oligonucleotide probes can also be applied for analysing biofilms or for the detection and identification of non-culturable bacteria. Moreover, they can be used to resolve individual target and non-target bacteria by flow cytometry.