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Titlecarranza's clinical periodontology
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(mRNA). Chromosomal DNA is dispersed in the bacterial cell without a membrane envelope.55
Diagnostic assays employing molecular biology techniques require specific DNA fragments that
recognize complementary-specific bacterial DNA sequences from target microorganisms.
Development of a microbiologic diagnostic test using this technology therefore requires the ability
to extract the bacterial DNA from the plaque sample and amplify the specific DNA sequence of the
target periodontal pathogens.

Nucleic Acid Probes

A probe is a known nucleic acid molecule (DNA or RNA) from a specific microorganism
artificially synthesized and labeled for its detection when placed with a plaque sample. DNA
probes use segments of a single-stranded nucleic acid, labeled with an enzyme or radioisotope that
is able to “hybridize” to the complementary nucleic acid sequence and thus detect the presence of
target microorganism. Hybridization refers to the pairing of complementary DNA strands to
produce a double-stranded nucleic acid. The nucleotide base-pair relationship is so specific that
strands cannot anneal unless the respective nucleotide strand sequences are complementary
(Figure 37-17). All hybridization methods use radiolabeled or fluorescence-labeled DNA probes
that bind to the target DNA of interest, thus allowing its visualization.* DNA probes may target
whole genomic DNA or individual genes. Whole genomic DNA is more likely to cross–react with
nontarget microorganisms because of the presence of homologous sequences between different
bacterial species. Currently, most of the probes used are oligonucleotides ranging from 20 to 30

Whole genomic probes for the detection of Aa, Pg, Pi, and Td have been developed and tested and
are the bases of commercially available diagnostic methods (e.g., DMDx, Omnigene). When
compared to culture, van Steenberghe et al.135 reported a sensitivity of 96% and specificity of 86%
for Aa and 60% and 82%, respectively, for Pg in pure laboratory isolates. However, when tested in
clinical specimens, both sensitivity and specificity were reduced significantly, suggesting cross-
reactivity with unknown bacteria in subgingival plaque samples. To overcome this drawback,
oligonucleotide probes complementary to variable regions of the 16S rRNA bacterial genes have
been developed for the detection of various periodontal pathogens. These bacterial 16S rRNA
genes contain both regions shared by different bacteria and short stretches of variable regions
shared only by specific organisms of the same species or genus.93 When these oligonuclotide
probes where compared with culture in clinical samples for the detection of Aa, Pg, and Pi, Savitt
et al.120 reported an effectiveness of 100% in detecting Aa and Pi and of 91% in detecting Pg,
calculated at culture–positive levels (103 cells). However, DNA probes were more sensitive than
culture in detecting these pathogens in samples from periodontitis patients; for example, Aa was
detected by probe analysis in 70% of localized aggressive (juvenile) periodontitis samples, but
only detected in 10% by culture analysis. Conversely, when these probes were compared with IFA
for the detection of Pg and Tf, IFA showed significantly higher detection rates and higher

Figure 37­17

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