ENDODONTOLOGY. 35. Molecular Diagnostic Methods in Endodontics. MOKSHA NAYAK * #. MITHRA N. HEDGE ** #. NANDA KISHORE K. J. *** #. * Professor ...
ENDODONTOLOGY Molecular Diagnostic Methods in Endodontics MOKSHA NAYAK * # MITHRA N. HEDGE ** # NANDA KISHORE K. J. *** #
ABSTRACT st
As we are marching towards 21 century there is gradual shift in exploring endodontic infections from cultural era to molecular era (phenotypic to genotypic). As high propositional of bacterial species in this planet is uncultivable & as endodontic microbiota is more diverse & far more complex then expected 40% of this microbiota are uncultivable, this has sparkled a revived interest in molecular diagnostic methods in diverse endodontic discipline.
Proper exploiting this molecular technology
INTRODUCTION The principle of bacterial cultivation and
to explore the composition of endodontics
identification is known as phenotypic methods.
microbiota provides valuable information regarding
These methods analyze readily observable bacterial
identification and better understanding of causative
traits and “behavior.” Although these strategies are
factor to enhance high success rate in endodontic
the mainstay of diagnostic bacteriology, notable
treatment.
limitations are associated with the use of this
Before knowing molecular diagnostic method
phenotypic method.
it is important to know the molecular biology of different microorganism.
Explosion in molecular biology over the past 20years has provided alternatives to phenotype-
Microorganisms are divided into:
based strategies used in clinical microbiology. These
1. Prokaryotes
alternatives have the potential to avert some of the aforementioned limitations. The detection and manipulation of nucleic acids (DNA and RNA) allows microbial genes to be examined directly (i.e., genotypic methods) rather than by analysis of their products such as enzymes (i.e., phenotypic
Does not have membrane bonded nuclei
No mitochondria & golgi complex present.
Among the latest molecular diagnostic
Eg. Bacteria and archaeal cells
methods, polymerase chain reaction (PCR) is a gold
These cells have 70 S Ribosome composed
methods).
standard.
of 30S & 50S
* Professor, ** Head of the department, *** PG Student. # Department of Conservative Dentistry and Endodontics, A.B.Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore.
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ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
30 S subunit contain 16S rRNA molecules
Genotypic identification involves characte-
approximately 1540 nucleotides
rization of some portion of bacterium’s genome
50S subunit contain 23S rRNA molecules
using molecular techniques for DNA or RNA
approximately 2900 nucleotides & 5Sr
analysis. This usually involves detecting the
RNA- 120 nucleotides
presence of a gene, or a part of thereof, or an RNA product that is specific for a particular organism. In
2. Eukaryotes
principle the presence of a specific gene or a particular nucleic acid sequence is interpreted as a definitive identification of the organism. The genotypic approach is highly specific and often very sensitive
OVERVIEW OF MOLECULAR METHODS
Membrane bounded nucleus Ex. Fungi
The molecular methods to be discussed are
Cells have 80 S ribosomes composed -40s
classified into one of three categories:
subunit and 60s subunit i. 40s subunit -18Sr RNA
1.
Hybridization
ii. 60s subunit - 25Sr RNA
2.
Amplification
- 5.8Sr RNA
3.
Sequencing
4.
Enzyme digestion of nucleic acid
Small subunit genes 16Sr DNA & 18S rDNA Larger subunit genes 23Sr DNA & 25Sr
1. Hybridization Methods
DNA have been widely used microbial
Hybridization methodology employes DNA
identification.
probes, single stranded DNA, labeled with an
3. ProtozMoa
enzyme, radioactive isotope or chemiluminescence
ORGANISM IDENTIFICATION USING GENOTYPIC CRITERIA
reporter, which locates and binds to complementary nucleic acids sequence of known identity To form a double-stranded molecule, or duplex or hybrid. This duplex formation is driven by the consistent manner in which the base adenine always bonds to thymine, while the bases guanine and cytosine always form a bonding pair. Because hybridization requires nucleic acid sequence homology, a positive hybridization reaction between two nucleic acid strands, each from a different source, indicates genetic relatedness between the two organisms that donated each of 36
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
the nucleic acid strands for the hybridization
of a probe depends on the intended use. For
reaction.
example, if a probe is to be used for recognizing only gram-positive bacteria, the probe’s nucleic acid
Hybridization assays require that one nucleic
sequence needs to be specifically complementary
acid strand (the probe) originates from an organism
to a nucleic acid sequence common only to gram-
of known identity and the other strand (the target)
positive bacteria and not to gram- negative bacteria.
originates from an unknown organism to be detected
Even more specific probes can be de signed to
or identified. Positive hybridization identifies the
identify a particular bacterial genus or species,
unknown organism as being the same as the probe-
virulence, or an antibiotic resistance gene that may
source organism. With a negative hybridization test,
only be present in certain strains within a given
the organism remains undetected or unidentified.
species.
The single-stranded nucleic acid components used in hybridization may be either RNA or DNA so that
In the past, probes were produced through a
DNA-DNA, DNA-RNA, and even RNA-RNA
labor- intensive process that involved recombinant
duplexes may form, depending on the specific
DNA and cloning techniques with the piece of
design of the hybridization assay.
nucleic acid of interest. More recently, probes are chemically synthesized using instrumentation, a
HYBRIDIZATION STEPS AND COMPONENTS
service that is widely available commercially.
The basic steps in a hybridization assay
2. Preparation of single-stranded target
include:
nucleic acid Generally, target preparation steps involve
1. Production and labeling of single-stranded
enzymatic and/or chemical destruction of the
probe nucleic acid
microbial envelope to release target nucleic acid, stabilization of target nucleic acid to preserve
2. Preparation of single-stranded target
structural integrity, and, if the target i DNA,
nucleic acid
denaturation to a single strand, which is necessary
3. Mixture and hybridization of target and
for binding to complementary probe nucleic acid.
probe nucleic acid
In Fungi – as the cell wall is thick need
4. Detection of hybridization
additional steps/process to get target nucleic acid
Steps
3. Mixture and hybridization of target and
1. Production and labeling of single-stranded
probe nucleic acid
probe nucleic acid In keeping with the requirement of
4. Detection of hybridization
complementation for hybridization, the probe
The method of detecting hybridization depends
design (i.e., probe length and its sequence of nucleic
on the reporter molecule used for labeling probe
acid bases) depends on the sequence of the intended
nucleic acid and on the hybridization format.
target nucleic acid. Therefore, selection and design
Detection of hybridization using radioactively 37
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
labeled probes is done by exposing the reaction
detracts from the potential speed advantage that
mixture to radiograph film (i.e., autoradiography).
molecular methods can offer. However, the
Hybridization with nonradioactively labeled probes
development of molecular amplification techniques
is detected using colorimetry, fluorescence, or
not dependent on organism multiplication has
chemiluminescence, and detection can be
contributed greatly to the circumvention of the
somewhat automated using spectrophotometers,
speed problem while enhancing sensitivity and
fluorometers, or luminometers, respectively. The
maintaining specificity. The three strategies for
more commonly used nonradioactive detection
molecular amplification are target nucleic acid
systems (e.g., digoxigenin, chemiluminescence) are
amplification, nucleic acid probe amplification, and
able to detect approximately 1O target nucleic acid
amplification of the probe “signal.”
sequences per hybridization reaction.
Coined by Kary Mullis- 1983
DNA HYBRIDIZATION
PCR is unique in its ability to locate and exponentianally amplify a small quantity of specific nucleotide sequence which is lost against a large back ground of nucleic acid. 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 in short time and then this amplified target formed is detected by different methods. Steps in PCR:
2. Amplification / PCR
1 Denaturation of target nucleicacid
Although hybridization methods are highly
2 Primer annealing
specific for organism detection and identification,
3 Extension of primer- target duplex /
they are limited by their sensitivity, that is, without
polymerization
sufficient target nucleic acid in the reaction, false1. Denaturation of target nucleicacid:
negative results occur. Therefore, many hybridization methods require “amplifying” target
- In which the DNA strands separate
nucleic acid by growing target organisms to greater
- Occurs at (94-950 C)
numbers in culture. The requirement for cultivation 38
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
STEPS IN PCR AMPLIFICATION
2 Primer annealing Primer – short sequence of olygonuclotides (20-30) nucleotide long is 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 0 C, and the polymerization occurs with the help of Tac polymerase enzyme. Thus at the end of one cycle, 2 duplex strand are formed, Each of which can again be denatured readily for the second cycle of hybridization and extension, thus at the end of second cycle four duplex stand (8 nucleotides are formed, thus at the end of 30 -40 cycles result in information of 107 -108 target copies.
NOTE
The optimal temperature for the annealing step will depend on primer used
The time taken for each cycle is considerable longer than 3mins,time taken for the overall reaction is 2 – 3 Hrs
PCR dose not efficiently amplify sequence much longer than about 3 Kb
Derivatives of PCR 1. Multiplex PCR 2. Nested PCR 3. Arbitary primed PCR 4. Quantitative PCR 5. Reverse Transcriptase PCR 6. Real Time PCR
39
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
1 .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
2. Cloning and Sequencing The amplified product is detected by help of automated DNA sequencer in with each type of dideoxynucleotide emits colored light of a characteristic wave length and is recorded as colored band
3.DGGE Analysis ( Denaturing Gradient Gel Electrophoreses) Detection of amplified PCR product
DGGEA technique is based on electrophoreses
1. Electrophoreseis in agrose gel
of PCR amplified fragment in polyacrylamide gel
2. Cloning and sequencing
containing linear increasing gradient of DNA
3. DGGE analysis ( denaturing gradient gel
denaturants
electrophoreses) 4. T- RFLP analysis ( terminal restriction fragment length polymerase analysis) 5. Micro array analysis 6. Reverse capture checker board analysis 40
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
1. DNA micro array analysis ( high density dna probe) In with high density oligonucleotide array (>500 million) is created using light directed chemical synthesize on a miniature glass substrate or chip, with is then hybridized to DNA, then the image processing soft ware analyses intensity of hybridization
REAL TIME PCR The most commonly employed derivative of PCR.Recently, a number of commercially available systems have been introduced that are capable of detecting the presence of a target within 30 to 120 minutes using PCR; the process by which detection is accomplished is referred to as “real time” PCR. Real time PCR combines rapid thermocycling with the ability to detect target by fluorescently labeled probes as the hybrids are formed, i.e., in real time. This technology allows for high throughput of
Limitations of PCR- derived technologies
samples, multiplexing reactions, quantitation of
a) Most PCR assays used for identification
target, and on-line monitoring.
purposes Is the most commonly employed method of
qualitatively
detect
the
target
microorganism but not its levels in the sample.
PCR for identification of microrganism
Quantitative results can however be obtained in real-time PCR assays.
In which the PCR product is detected in real time, by using fluorescent labeled probe:
b) Most PCR assays only detect one species or
- Taq Man probe
a few different species (multiplex PCR) at a time.
- Molecular becon
However, broad-range PCR analysis can provide
- Sybar green
information about the identity of virtually all species in a community.
Taq Man probe 41
ENDODONTOLOGY
MOLECULAR DIAGNOSTIC METHODS IN ENDODONTICS
c) Like DNA-DNA hybridization, most PCR
organisms (for instance Tannerella forysthesis,
assays only detect target species and consequently
Trepenoma denticoli, other Trepenoma species,
fail to detect unexpected species. This can be
Dialister pneumositis, Prevotella tanneria were
overcome by broad-range PCR assays.
detected in the infected root canal for the first time by using PCR analysis ).
d) In addition to being laborious and costly, broad-range PCR analyses can be affected by some
Despite of great advance and revolutions
factors, such as biases in homogenization
bought about by molecular diagnostic method in
procedures (76), preferential DNA amplification
diverse medical discipline, endodontic microbiota
(112, 113) and differential DNA extraction.
is still undergoing shift from cultural era to molecular era, proper exploiting this molecular diagnostic
e) Microorganisms with thick cell walls, such
method in endodontics enhance high success rate
as fungi, may be difficult to break open and may
in endodontic treatment.
require additional steps for lysis and consequent
References
DNA release to occur.
1. Baily and Scott: molecular methods of microbial identification and characterization; Diagnostic microbiology 11th edition, 169-188
f) False positive results have the potential to occur because of PCR amplification of contaminant
2. E.D.P.De Robertis. Cell and molecular biology. 8th edition.
DNA. The most important means of contamination
3. Lisa Aunishimeld &Aune T.Rodgers .Essentials of diagnostic microbiology.
is through carryover of amplification product and special precautions should be taken to avoid this
4. David Greenwood & Richard C.B.Slack.Medical microbiology. 16th edition
g) False negatives may occur because of
5. Siqueira JF. Exploiting molecular method to explore endodontic infections: Part 1 – Current Molecular Technologies for Microbiological Diagnosis. Journal of Endodontics, 2005; 31(6): 411-423.
enzyme inhibitors or nucleases present in clinical samples, which may abort the amplification reaction and degrade the DNA template, respectively.
6. Siqueira JF. Exploiting molecular method to explore endodontic infections: Part 2 – Current Molecular Technologies for Microbiological Diagnosis. Journal of Endodontics, 2005;31(7) : 488-497.
Analysis of small sample volumes may also lead to false negative results, particularly if the target species is present in low numbers.
7. Siqueira JF.PCR methodology as a valuable tool for identification of endodontic pathogens. Journal of Dentistry, 2003; 31: 333-339.
CONCLUSION Culture methods have provided a great
8. Becker W, Becker B, Newman M, Nyman S. Clinical and microbiologic findings that may contribute to dental implant failure. Int J Oral Maxillofac Impl. 1990: 5: 31—38.
contribution to, and has still much to offer in elucidation of endodontic disease. However molecular approaches to detect and identify
9. Rocas IN, Jung Y, Lee CY & Siqueira JF. Polymerase Chain Reaction Identification of Microorganisms in Previously RootFilled Teeth in a South Korean Population. Journal of Endodontics, 2004; 30:504-508.
microbial species have several advantages when compared with culture.
Oliveira JCM, Siqueira JF. Detection of Porphromonas endodontalis in Infected Root Canals by 16S rRNA GeneDirected Polmerase Chain Reaction. Journal of Endodontics,2000; 26:729-732.
Molecular method, particularly PCR are more specific, accurate, sensitive and rapid than culture and can detect uncultivable and fastidious micro 42