There has been a remarkable improvement in microbiological research in periodontics. The most basic approach is culture-dependent approach. Culture-dependent approaches are extremely useful for determining the antibiotic susceptibility of oral microbes and for assessing the pathogenicity of individual species. Periodontal plaque sample is allowed to grow in specific medias where the colonies of different species of micro-organisms can be identified. Disadvantage is that it requires experienced well trained labs and it is time consuming.
Presently we have culture independent approaches which involve RNA and DNA sequence identification techniques. Basically there are three main categories of molecular microbial analyses which include
- Polymerase chain reaction (PCR)-based methods (single target PCR, multiplex PCR and quantitative PCR),
- DNA–DNA hybridization methods (in situ hybridization, checkerboard hybridization and 16S ribosomal RNA-based microarrays) and
- Sequencing methods (latest, next generation sequencing techniques, such as pyrosequencing, real-time single-molecule DNA sequencing and nanopore-based sequencing).
Here is a brief description of currently used molecular microbial techniques
16S ribosomal RNA-based, reverse-capture oligonucleotide probes:
These are used to identify the microorganism species in plaque samples. 16S ribosomal RNA-based microarray is currently commercially available for diagnostic use. The ParoCheck® (Greiner bio-one, Frickenhausen, Germany) DNA chip targets 20 oral bacterial species and has been used to determine the microbial profiles of clinical samples. Recently high-density 16S ribosomal RNA-based microarrays have also been developed. The Phylochip, developed by the Affymetrix Corporation® (Santa Clara, CA) and Lawrence Berkeley Laboratories (Berkeley, CA) can detect up to 32,000 16S ribosomal RNA phylotypes1-3.
DNA Checkerboard hybridization:
This procedure involves extracting DNA from oral samples and hybridizing the sample against labeled probes representing whole genomes or 16S ribosomal RNA genes of known microbes. The method can be used to identify the subgingival plaque species in periodontal health and disease4-7.
Fingerprinting of amplified PCR products:
This method involves the digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer8-10.
This method is used for quantifying the bacteria or genes in a given microbial plaque sample11-14.
It is based on the principal of sequence of enzyme triggered reactions that ultimately leads to the release of a pyrophosphate which in turn leads to generation of a luminescence signal after successful incorporation of a nucleotide into the DNA sequence15-16.
They contain oligonucleotide probes that target RNA and DNA genes in biological samples17-20.
Research in any of above requires high end microbiological labs with highly trained personal which can give you accurate results without any error. A lot of gene based studies have been done. A thorough knowledge about the procedures involved is required before starting the study.
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