MOLECULAR DIAGNOSTIC

AIDS IN ENDODONTICS
ANISHA
Introduction

culture methods

Molecular biology techniques

Gene targets for microbial identification

PCR and its derivatives

Denaturing gradient gel electrophoresis

Terminal restriction fragment length polymorphism

DNA–DNA hybridization assays

Fluorescence in situ hybridization

Advantages and limitations of molecular methods

Bacterial diversity in the oral cavity

Primary intraradicular infections

Persistent/secondary intraradicular infections

Extraradicular infections

Conclusion
Introduction

Accurate and definitive microorganism identification, including

bacterial identification and pathogen detection, is essential for
correct disease diagnosis, treatment of infection.
Conventional culture methods

Culture is the process of propagating

microorganisms in the laboratory

by providing them with proper environmental

conditions.

Essentially culture analysis involve the following

steps:
Advantages

1. Broad-range nature, identification of unexpected species

2. Allow quantification of all major viable microorganisms

3. Allow determination of antimicrobial susceptibilities of the isolates

4. Physiological studies are possible

5. Pathogenicity studies are possible

6. Widely available
Limitations

1. Impossibility of culturing a large number of microbial species

2. Not all viable microorganisms can be recovered

3. Once isolated, microorganisms require number of techniques

4. Misidentification of strains

5. Low sensitivity

6. Strict dependence on the mode of sample transport

7. Samples require immediate processing

8. Costly, time consuming and laborious

9. Specificity is dependent on the composition of media

10. Extensive expertise and specialized equipment

11. It takes several days to weeks to identify most anaerobic

bacteria
Why molecular methods

diagnostic methods should be more site-specific and accurate than

the tests we currently have.

avoid overtreatment and to maintain vital tissues

Bacteria or host response?

Targeting bacteria allows direct assessment of the cause of disease.

However, endodontic infections --- non-specific and composed of

mixed microorganisms
In the context of molecular diagnostics, there are some clear
advantages to assess the host response rather than bacterial invasion

Each site and tissue fluid has its specific environment of host factors

The composition of the respective analyte can be altered because of

inflammatory process.
Which fluid to collect

Gingival crevicular fluid (GCF)

GCF is an exudate that derives from the gingival crevice.

studies showed that the composition of neurotransmitters, IL -8 or

MMP-8 differs in GCF of painful and non-painful teeth .
Drawbacks.

Impossible to distinguish between a marginal and an apical periodontal

inflammation.

bias from gingival or periodontal inflammation in GCF analyses

GCF is unlikely to be able to reflect ---- level of microbial progress in

the pulp space
Pulpal blood
Dr. Florian Prader at the University of Zürich in 1949

he compared pulpal blood smears of teeth with advancing endodontic

infection --- elevation of cellular blood compounds (PMNs).

15 yrs later, Guthrie and co-workers published their classic study on

pulpal blood examination.

This study highlighted again the pivotal role of PMNs in the breakdown of the pulp.
Nakanishi et al ----- IgG, IgA, IgM, elastase, and especially
prostaglandin E2,

IL-1 and IL-6 were elevated in inflamed compared to healthy teeth.

neutrophil-attracting chemokine IL-8 was significantly elevated in

symptomatic pulpitis
Drawbacks

sampling pulpal blood requires entering the pulp space

blood interferes with most protein assays

Dentinal fluid

Dentinal fluid is the extracellular fluid that is contained in dentinal

tubules

MMP-9 was identified by a highly sensitive assay in 7 of 16 teeth

diagnosed with irreversible pulpitis
Periapical fluid

The term “periapical fluid”, or “periapical tissue fluid” refers to the

extracellular fluid present in the periapical area.

exudate formation is an immediate host reaction,

analysis of the exudate --- current state of inflammation
analyzing periapical fluid --- inflammatory response to a microbial
infection in a closed environment

Bacteria-related factors like endotoxin, and host-related factors like

cytokines, immunoglobulins, or MMPs have been identified from
periapical exudates

Periapical fluid could be especially helpful to monitor early signs of

healing
Which target molecules should be considered?

Histologic studies clearly show that pulpitis is a PMN-driven

inflammation

PMN-related enzymes such as Elastase and Cathepsin G and MMP-9

are elevated in clinically inflamed compared to healthy dental pulps.

IL-8, the main neutrophil chemoattractant.

OVERVIEW OF MOLECULAR METHODS

The molecular methods to be discussed are classified into one of three

categories:
1. Hybridization
2. Amplification
3. Sequencing
4. Enzyme digestion of nucleic acid
1.Hybridization Methods

Hybridization methodology employes DNA probes.

single stranded DNA, labeled with an enzyme, radioactive isotope or

chemiluminescence reporter, which locates and binds to
complementary nucleic acids sequence of known identity
HYBRIDIZATION STEPS AND COMPONENTS

The basic steps in a hybridization assay include:

1. Production and labeling of single-stranded probe nucleic acid

2. Preparation of single-stranded target nucleic acid

3. Mixture and hybridization of target and probe nucleic acid

4. Detection of hybridization
1. Production and labeling of single-stranded probe nucleic acid

depends on the intended use.

Ex : if a probe is to be used for recognizing only gram +ve bacteria, the

probe’s nucleic acid sequence -- specifically complementary to a nucleic
acid sequence common only to gram +ve bacteria
2. Preparation of single-stranded target nucleic acid

enzymatic and/or chemical destruction of the microbial envelope to

release target nucleic acid

stabilization of target nucleic acid to preserve structural integrity

denaturation to a single strand

• 3. Mixture and hybridization of target and
probe nucleic acid
4. Detection of hybridization

Detection of hybridization using radioactively labeled probes is done

by exposing the reaction mixture to radiograph film
(autoradiography).

Non radioactively labeled probes ---- colorimetry, fluorescence, or

chemiluminescence
DNA- DNA Hybridization assays

2 types

Checker board DNA-DNA hybridization

DNA micro arrays

Checker board DNA-DNA hybridization

Socransky et al in 1994
hybridizing large no of DNA samples against large numbers of
digoxigenin-labeled whole genomic DNA on a single support membrane

denatured DNA from clinical samples --- nylon membrane using a

Minislot apparatus. After fixation of the samples to the membrane ----
placed in a Miniblotter 45 apparatus with the lanes of samples at 90◦ to
the lanes of the device.
Digoxigenin-labeled whole genomic DNA probes are then loaded.

After hybridization, the membranes are washed --- DNA probes detected using
antibody to digoxigenin conjugated with ALP and chemifluorescence detection.

The presence of a spot ---- hybridization has occurred.

The intensity of the spot is proportional to the amount of DNA from the target
species
A modification of the checkerboard method --- Paster et al. (1998)

In this assay, the probe rather than the sample is fixed first to the
membrane

— reverse-capture checkerboard.
DNA Microarrays -- Schena et al in 1995

highdensity matrix of DNA probes, which are printed or synthesized on a glass

or silicon slide (Mothershed and Whitney 2006)

Targets incorporate a fluorescent label --- hybridization --- detected using

reporter molecule.

Following hybridization, arrays are imaged -- high-resolution laser scanner --

software analysis

used to enhance PCR

Fluorescence insitu hybridization (FISH)

uses fluorescently labeled rRNA directed probes and fluorescence

microscopy to detect intact microbial cells directly in clinical specimens, in
situ (Moter and Gobel 2000)

Adv :

identification and provide information about morphology, number,

community architecture and spatial relationship of microorganisms.
FISH protocol

Fixation and permeabilization of the sample

Hybridization with the respective probes

Washing to remove unbound probe

Detection of labeled cells by microscopy or flow cytometry

2. Amplification / PCR

hybridization methods require “amplifying” target nucleic acid by

growing target organisms to greater numbers in culture.

3 strategies for molecular amplification are

target nucleic acid amplification,

nucleic acid probe amplification, and
amplification of the probe “signal.”
PCR is unique in its ability to locate and exponentianally amplify a small
quantity of specific nucleotide

Uses the principle of nucleic acid hybridization and nucleic acid

replication & apply repeatedly for number of cycle.

Single copy of nucleic acid which is not detected by hybridization

method is multiplied to 10 * 7 – 10 * 8 more copies by 30 -50 repetitive
cycle
Steps in PCR:

1 Denaturation of target nucleicacid

2 Primer annealing
3 Extension of primer- target duplex / polymerization
1. Denaturation of target nucleicacid:

The target DNA serving as template is denatured (melted)

at temperatures high enough to break the hydrogen

---- single strands of DNA.

Occurs at (94-950 C)
2. Primer annealing

Primer -- short sequence of olygonuclotides --- hybridized (annealed)

to particular nuclicacid target which act as a probe result in duplex
formation

Occurs at (55 –650 C )

3. Extension of primer- target duplex / polymerization

Occurs a 72 C

the polymerization occurs with the help of Tac polymerase enzyme.

at the end of one cycle, 2 duplex strand are formed

PCR is 10- to 100-fold more sensitive (Siqueira and Rˆoc¸as 2003)

Derivatives of PCR

Species specific PCR

Multiplex PCR
Nested PCR
Arbitary primed PCR
Quantitative PCR
Reverse Transcriptase PCR
Real Time PCR
Species-specific PCR

primers designed to anneal signature genomic sequences of a given

species are used to detect this species directly in clinical samples

the variable regions of the 16S rRNA gene to design primers specific
for bacterial species.
Multiplex PCR

Multiplex PCR is a process where multiple primer

pairs are used to simultaneously amplify several sequences
in a single reaction (Chamberlain et al.)

more than one unique target sequence -- amplified at the same time,

Multiplex PCR assays permit the concomitant detection

of different species
Nested PCR

that amplifies a target region of DNA with an outer

primer pair in an initial reaction, followed by

a second amplification using an internal primer pair (Haqqi et al)

The first PCR products are used as template in the second round of
amplification with a separate primer set

---- shorter amplified fragment.

Reverse transcriptase-PCR

Developed to amplify RNA targets and exploits the use of the

enzyme reverse transcriptase, which can synthesize a
strand of cDNA from RNA template.

Most RT-PCR assays employ a two-step approach.

1st step,
reverse transcriptase converts RNA into single-stranded cDNA.

2nd step,
PCR primers, DNA polymerase, and nucleotides are added to create
the second strand of cDNA.

Once the double-stranded DNA is formed, it can be used

as template for amplification as in conventional PCR
(Sambrook and Russell 2001)
Quantitative PCR

Conventional PCR assays are qualitative or semi-quantitative.

Basically, quantitative PCR (qPCR) can be performed using 3 assays:

most probable number (MPN)-PCR,

competitive PCR,
Realtime PCR (Sharma et al. 2007).
REAL TIME PCR

The most commonly employed derivative of PCR

30 to 120 minutes

Real time PCR combines rapid thermocycling with the ability to detect
target by fluorescently labeled probes as the hybrids are formed

This technology allows for high output of samples, multiplexing

reactions, quantification of target, and on-line monitoring.
fluorescent labeled probe:

• Taq Man probe

• Molecular becon
• Sybar green
Taq Man probe

TaqMan assay results from the utilization of a specific labeled

oligonucleotide probe along with the primers

The TaqMan probe contains a reporter fluorescent dye at the 5’ end and a
quencher dye at the 3’ end

As long as the probe remains unbound -- no signal

During the extension real-time PCR, Taq DNA polymerase enzyme cleaves
the TaqMan probe, resulting in separation of the reporter from the
quencher.
---- fluorescence emission.
Molecular beacons

are single-stranded nucleic acid molecules -- stem-and-loop structure

(Mackay 2004)

The loop portion is complementary to a sequence in the target DNA.

The stem is formed by the annealing of complementary arm sequences
located on either side of the probe.
A fluorescent marker is attached to the end of one arm, and a quencher
is attached to the end of the other arm.

Hybridization their complementary target, molecular beacons

--- conformational change

The conformational change forces the fluorescent dye and the quencher
apart --- fluorescence (Mhlanga and Malmberg 2001)
SYBR Green

consists of a fluorescent dye that binds to double stranded DNA.

During extension, increasing amounts of dye bind to the increasing

amount of newly formed double-stranded DNA
Detection of amplified PCR product

1. Electrophoreseis in agrose gel

2. Cloning and sequencing
3. DGGE analysis ( denaturing gradient gel electrophoreses)
4. T- RFLP analysis ( terminal restriction fragment length polymerase
analysis)
5. Micro array analysis
6. Reverse capture checker board analysis
Electrophoresis In Agrose Gel

The amplified product is subjected to agrose gel stained with

ethidium bromide

The amplified DNA fragment is separated by agrose gel using

molecular weight size of each amplicon (Large sizemoves less,
Smallsize –moves fast)

This separated amplicon glows when illuminated with U V light

Cloning and Sequencing

The amplified product is detected by help of automated DNA

sequencer

each type of dideoxynucleotide emits colored light of a

characteristic wave length and is recorded as colored band
DGGE Analysis

( Denaturing Gradient Gel Electrophoreses)

electrophoreses of PCR amplified fragment in polyacrylamide gel

containing linear increasing gradient of DNA denaturants
DNA micro array analysis

( high density DNA probe)

high density oligonucleotide array (>500 million) is created on a

miniature glass substrate or chip

with is then hybridized to DNA

software analyses ---- intensity of hybridization

Terminal restriction fragment length polymorphism (T-RFLP)

One of the PCR primers is labeled with a fluorescent dye.

PCR amplicons are then digested with restriction enzymes,

generating fluorescent labeled fragments of different lengths

-----(Terminal fragments)
These fragments are separated on
high resolution sequencing gels in an
automated DNA sequencer
SEQUENCING

The amplified product is detected by help of automated DNA sequencer in which

each type of deoxynucleotide emits colored light of a characteristic wave length

an recorded as colored band

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PYROSEQUENCING
Method of DNA sequencing that relies on the detection
of pyrophosphate release on nucleotide incorporation.

The desired DNA sequence is determined by light emitted upon

incorporation of complementary nucleotide by the fact that only one out
of four of the possible A/T/C/G nucleotides are added and available at a
time
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ADVANTAGES OF MOLECULAR METHODS

Detect both cultivable and as yet cultivated species

High specificity and accurate identification of strains with ambiguous

phenotypic behavior

Detect species directly in clinical samples

High sensitivity

Rapid- most assays take no more than a minutes to a few hours to

identify a microbial species
68
 LIMITATIONS OF MOLECULAR METHODS

Whole genomic probes used in the DNA-DNA hybridization assays may

show cross-reactivity with non target micro-organisms.

This can give rise to false positive results.

69
Limitations of PCR- derived technologies

a) Most PCR assays used for identification purposes qualitatively

detect the target microorganism but not its levels in the sample

b) Most PCR assays only detect one species or a few different

species (multiplex PCR) at a time

c) Like DNA-DNA hybridization, most PCR assays only detect target

species and consequently fail to detect unexpected species
d) In addition to being laborious and costly, broad-range PCR analyses can
be affected by biases in homogenization procedures, preferential DNA
amplification and differential DNA extraction.

e) Microorganisms with thick cell walls, such as fungi, may be difficult to

break open and may require additional steps for lysis and consequent DNA
release to occur

f) False positive results have the potential to occur because of PCR

amplification of contaminant DNA.
g) False negatives may occur because of enzyme inhibitors or
nucleases present in clinical samples, which may abort the
amplification reaction and degrade the DNA template, respectively.

Analysis of small sample volumes may also lead to false negative

results, particularly if the target species is present in low numbers.
The “dead cell issue”

Detection of dead cells

this allows detection of uncultivable bacteria / fastidious bacteria that can

die during sampling, transportation or isolation procedures

On the other hand, if the bacteria were already dead in the infected site

--------- false assumption about their role in the infectious process

Bacterial diversity in the oral cavity
13 separate phyla.

The majority of oral species/phylotypes belong to the phyla

Firmicutes
Fusobacteria
Bacteroidetes
Actinobacteria
Proteobacteria
Spirochaetes
Synergistes (Lillo et al. 2006; Aas et al. 2007) 74
Caries

Streptococcus, Lactobacillus, and Actinomyces -- etiopathogenesis

of different forms and stages of caries
(Marsh and Martin 1999; Bowden 2000).

]species of Streptococcus, Lactobacillus, and Actinomyces with

caries, uncultivated phylotypes of Bifidobacterium and Atopobium
have also been suggested
(Becker et al. 2002; Aas et al. 2003)

75
As the caries lesion advances deep into dentin,

microbiota shifts from a predominance of facultative and

saccharolytic Gram positive bacteria in shallow lesions

lactobacilli and/or proteolytic anaerobic bacteria

(Martin et al. 2002; Chhour et al. 2005)

76
Other taxa----

Selenomonas
Dialister
Fusobacterium
Eubacterium
Bifidobacterium
(Chhour et al. 2005)

77
Periodontal diseases
Periodontal diseases result from the subgingival presence
of complex bacterial biofilms

Analysis of the subgingival bacterial communities--- checkerboard

approach --- 5 major bacterial complexes (Socransky et al. 1998).

The most pathogenic complex (red complex) comprised the cultivated

species

T. forsythia, P. gingivalis, and Treponema denticola

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Others

Dialister pneumosintes, Dialister invisus,

Filifactor alocis,

Prevotella denticola,

Cryptobacterium curtum,

Treponema medium, Treponema socranskii,

Eubacterium saphenum,

Catonella morbi, and

Selenomonas sputigena

(Doan et al. 2000; Paster et al. 2001; Kumar et al. 2003, 2005). 79
Microbiota in root canal–treated teeth

Gram-positive bacteria with Enterococcus faecalis

P. alactolyticus, and P. propionicum

E. faecalis --- prevalence 90% of the cases --- Williams et al. 2006
Molecular analyses of extraradicular Infection

Fusobacterium nucleatum,
Peptococcus micros,
Actinomyces israelii, Actinomyces viscosus,
Campylobacter rectus,
P. gingivalis,
Tannerella forsythia,
Treponema socranskii ssp. socranskii,
Aggregatibacter actinomycetemcomitans,
Streptococcus spp.
--------Was present in more than 70% of the lesions 82
Other designated endodontopathogens such as

Treponema denticola (60%),

A. odontolyticus (50%),
P. endodontalis (50%),
Eikenella corrodens(40%) were commonly present.

In 20–30% of the lesions,

Campylobacter gracilis, Eubacterium nodatum.

83
Other microorganisms in endodontic infections

Fungi

species-specific PCR -- Candida albicans in 21% -- primary root

canal infections
(Baumgartner et al. 2000).

9% of the root canal–treated teeth with apical periodontitis

(Siqueira and Rˆoc¸as 2004b).

Archaea

methanogenic archaea in 25% of the canals -- untreated teeth with

chronic apical periodontitis (Vianna et al. 2006a)

Archaeal diversity was limited to a Methanobrevibacter oralis–like

phylotype
Virus

RT-PCR -- HCMV , EBV ----in apical periodontitis lesions,

where living host cells abound.

HCMV and EBV transcripts -- symptomatic apical periodontitis lesions

in lesions exhibiting elevated occurrence of anaerobic bacteria
in cases of large periradicular bone destruction

(Sabeti et al. 2003b; Sabeti and Slots 2004)

Conclusion

Molecular method, particularly PCR are more specific, accurate,

sensitive and rapid than culture and can detect uncultivable micro
organisms

(for instance Tannerella forysthesis, Trepenoma denticoli, other

Trepenoma species, Dialister pneumositis, Prevotella tanneria were
detected in the infected root canal for the first time by using PCR
analysis ).
Undoubtedly, a great deal of additional research is needed to define
the specific role played by suspected endodontic pathogens in the
etiology of each form of periradicular disease and to determine the
best therapeutic measures for the pathogen’s eradication
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