Molecular Diagnostic Methods

The advent of molecular genetic methods has revolutionized life sciences as a whole. It has its share of impact on microbiology in general and on oral and endodontic microbiology in particular. Molecular diagnostic methods for micro-organisms have several advantages over cultivation methods:

1) They can detect both cultivable and uncultivable microbial species and strains.

2) They have greater specificity.

3) They can detect microbial species directly in clinical samples, without the need for microbial.

4) They are more sensitive than other identification methods.

5) They are usually less time-consuming.

6) They do not require carefully controlled anaerobic conditions during sampling and transportations.

7) They can be used during antimicrobial treatment.

The molecular detection methods available are:

• DNA-DNA hybridization methodology — these methods use DNA probes.
• Polymerase Chain Reaction Method — It involves invitro replication of DNA and has often been referred to as the “Genetic Xeroxing”. A lot of variations of PCR are now available, such as Single PCR, Reverse Transcriptase (RT) PCR, Nested PCR, Real-Time PCR.

Molecular techniques have been used to detect bacteria in endodontic infections using oligonucleotide probes and checkerboard DNA-DNA hybridization analysis.

The checkerboard DNA-DNA hybridization has the following advantages:

• It permits the simultaneous determination of the presence of a multitude of bacterial species in single or multiple clinical samples.
• The method does not require bacterial viability and is particularly applicable in epidemiologic research.
• DNA-DNA hybridization technology has the additional advantage that DNA is not amplified. Thus, microbial contaminants are not cultured, nor are their DNA amplified.

However, the use of specific DNA probes limits the boundaries of the detection technique:

• As it assumes that these probes target the species of importance.
• There are inherent problems with checkerboard analysis, which stem from the lack of specificity of the whole genomic probes used. The technique cannot be used to determine the true diversity of potential pathogens from infected root canals.

Whilst molecular methods greatly facilitate identification of culture-difficult species and enhance the precision of taxonomic grouping, it is important to recognize the limitations of PCR-based methodology.

1) 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.

2) 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.

Molecular diagnostic method have enabled the detection of some cultivable species in infected root canal or periradicular abscess in higher prevalence values than ever reported by cultural studies. These include Porphyromonas endodontalis, Porphyromonas gingivalis, Fusobacterium nucleatum, Proprionibacterium propionicum, Actinomyces sp.

Molecular diagnostic methods have also expanded the list of putative endodontic pathogens by including some fastidious bacterial species or even uncultivable bacteria. T. forsythia, which has never been detected from infected root canal by cultivation method.