An improved systematic structure has been made
possible with the application of molecular tools to
obtain data from 16S rRNA gene sequences,
which allows enhanced differentiation between micro-organisms and led to the establishment of new genera
and species. During the last decade, molecular
techniques have been used for microbial identification
of root canal samples. Many of the species that are
reported as new are split off from previously established
genera and species, but the ease of identifying culture-difficult species and the specificity of PCR-based
methods has meant that some additional species can be
included as typical of the microbial flora of the infected
root canal. These include spirochaetes,
and the species Tanerella forsythensis (formerly Bacteroides
forsythus),
which are prevalent in infected root
canals yet difficult to cultivate.
Whilst molecular methods greatly facilitate
identification of culture-difficult species and enhance
the precision of taxonomic grouping, it is important to
recognize the limits as well as the contributions of PCR-based methodology. The high sensitivity of this method
implies that it is essential that contamination controls
be strictly applied, as contaminants may be easily
picked up in the sample and amplified by PCR. The
PCR technique is based on recognition of gene
sequences — not recovery of cultivable cells capable of
growth — so the main drawback of PCR-based
methods is that it may detect both living and dead
bacteria. Because DNA that persists after cell death
may be detected by PCR, the findings from root canal
samples may reveal more than just active contributors
but could also reflect a historical record of the micro-organisms that have entered and not survived in the
root canal. Culture-based methods also have their
limitations, which include a high degree of skill, labour and time for identification of species and that some
species are culture difficult or impossible to culture in
vitro. These issues are discussed more fully elsewhere,
but it is fair to say that both culture and molecular
methods each specifically contribute to the study of the
root canal flora.