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Applications of PCR in DiagnosticApplications of PCR in DiagnosticMedicineMedicine
Done by
Mohamed Khalid 09B212
Rajathilagam 09B213
Rajeeva Lochan 09B214
Sasi Devi 09B215
Sridhar 09B219
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OutlineOutline
Introduction
Need for new diagnostic methods
Terms frequently used in diagnosis
Molecular Diagnosis (PCR-based)
Advantages and pitfalls of PCR as a diagnostic tool
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Need for new diagnostic methods
Microscopy gives false positive results -
- T.vaginalis, N.gonorrhoeae Intracellular pathogens viruses, M.genitalium
Low sensitivity Chlamydia sp.,Neisseria sp. Seropositivity is common Chlamydia sp. Subtyping is mandatory HSV, HPV, HCV Microbial growth is slow M. Tuberculosis False negative results in patients receiving
antimicrobials Bacterial meningitis
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Terms frequently used in diagnosis
Diagnostic sensitivity is defined by thepercentage of persons who have the disorder ofinterest who have positive results on the assay.
Diagnostic specificity is defined by thepercentage of persons who do not have the
condition of interest who have negative resultson the assay.
Note:- Do not confuse diagnostic & analyticalparameters
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The analytical sensitivity of an assay represents the smallest amount of asubstance that can be accurately measured in a biological sample;analytical specificity is the assay's ability to measure a particular organismor substance, rather than another, in a sample. These characteristics are
distinct from diagnostic sensitivity and specificity. In the clinical setting,diagnostic sensitivity is defined by the percentage of persons who have thedisorder of interest who have positive results on the assay. Although onemight expect that an analytically sensitive assay should more readilyidentify those persons, the ability to measure a very small quantity of asubstance does not always translate into high diagnostic sensitivity. This
apparent contradiction results from the shortcomings of sampling a verysmall volume, variations in the clinical spectrum of disease, and possibledifficulties with specimen preparation and technical performance of theassay.
Diagnostic specificity is defined by the percentage of persons who do not
have the condition of interest who have negative results on the assay.False-positive reactions diminish the diagnostic specificity; these reactionsmay be particularly likely to occur in molecular assays as a result ofcontamination with amplified material from other reactions (carryover).
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Detection Of Pathogens
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Molecular Diagnosis
Polymerase chain reaction (PCR) is the best-known
and most successfully implemented diagnostic
molecular technology to date. It can detect slow-
growing, difficult-to-cultivate, or uncultivatablemicroorganisms and can be used in situations in
which clinical microbiology diagnostic procedures
are inadequate, time-consuming, difficult,
expensive, or hazardous to laboratory staff.
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PCR diagnosis can be used to detect a specific
pathogen or for identification of classes of
pathogens. To detect a specific pathogen, say Chlamydia
trachomatis, primers specific for C.trachomatis are
used and checked for amplification
To identify a broad range of pathogens, say all
bacteria, primers specific for 16S rRNA gene
(evolutionarily conserved in all bacterial species)
are used and checked for amplifiaction
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Viability assesment using PCR
Nucleic acid detection assays is not a reliable
indicator for assesing pathogen viability.
If pathogen viability is to be assessed , PCRanalysis at the RNA level provides information
about transcription activity of the organism, which
is a marker of viability.
Limitation:- can't be used for RNA viruses
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Biomarkers for diseases
Biomarkers or gene signatures are genes which are
specifically induced during disease and can identify a
disease process with high confidence.
For example , cytokines or chemokines and theirtranscription factors and receptors are selectively induced
and can be used to detect an infectious disease process
without the presence of pathogen itself.
Additionally , pathogen specific genes encoding for anti-microbial resistance or toxins can be used as diagnostic
biomarkers
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Single pathogen detection vs. Panel
strategy
Selecting a single PCR assay greatly relies on the
ability of the clinicians to indicate the suspected
organism , is a hit-or-miss approach and carries a
risk of misdiagnosing the patient Thus instead of selecting a single suspect pathogen
the clinician selects a syndrome-specific panel of
PCR assays that broadly covers all the potential
pathogens , greatly increasing it's diagnostic power
(multiplex pcr)
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Microbial pathogen Tissue submission Target gene
Anaplasma phagocytophila Whole blood 16S rRNA
Corynebacterium tuberculosisAspirate from abcess Phospholipase D exotoxin
Equine herpes virus 1 NPS , whole blood , TTW ,BAL , CSF
Glycoprotein B
Sarcosystis neurona CSF 18S rRNA
West nile virus Whole blood , CSF Envelope gene
Lawsonia intracellularis Feces 16S rRNA
Neospora hughesi CSF 18S rRNA
Equine influenza virus NPS , TTW , BAL Haemagglutinin
Examples of PCR assays for diagnosis
of infectious diseases
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Limitations of PCR assay diagnosis
FALSE POSITIVES :-
Arises due to background contamination due to exogenous
sources of DNA , mostly carry over DNA.
Contamination is more pronounced in those assays which useuniversal primers those targetting 16S rRNA gene.
None of the methods(UV treatment , enzyme digestion
,chemical treatment) has been shown to be entirely effective
without significant diminution of assay sensitivitySelf contained micro-chip platforms hold promise for best
means of decontamination and overall assay efficiency.
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False negatives :-
May be due to two reasons Relatively small sample volume permissible for
PCR reactions
Problems associated with PCR processing
The problems can be encountered by optimising the
concentration of target DNA for PCR reaction and
by monitoring the presence of any PCR inhibitors
and inducing various chaotropic , enzymatic andthermal methods of cell lysis to effectively liberate
microbial DNA content
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Limited detection space for
characterising detected pathogen
While PCR product detection and analysis havetypically been achieved using gel-electrophoresisand sequencing techniques, these approaches arelaborious and timeconsuming, which detracts fromclinical applicability.
Even real-time PCR (unfortunately!) has alimited ability to spectrally differentiate multiplefluorescent signals.
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Other novel approaches to analyse the
amplified products
MICROARRAY TECHNOLOGYMICROARRAY TECHNOLOGY:-DNA microarraysare constructed by spatiallyisolating specific
genome sequences to prearranged areas on a microchip.Flourescently labelled amplificationproducts arethen allowed to anneal to complementary sequences on the
chip, and the resultant pattern is spectrally analysed. The
main advantage of using microarrays for pathogen
detection is the potentially large number of target
sequences the system can discriminate simultaneously. Theuse of microarray technology for pathogen detection is still
in the development phase however.
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MASS SPECTROMETRYMASS SPECTROMETRY:-
In MALDI-TOF-MS, the organic moleculefor
example an amplified productis ionised andsubsequently identified based on its mass-to-charge
ratio .The advantagesof MALDI-TOF-MS lie in theinherent accuracy and the high-speed (one second)
of signal acquisition, making this technology anattractive candidate for high-throughput DNA
analysis.
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References
Samuel Yang and Richard E Rothman(2004)PCRbased diagnostics for infectious diseases:uses, limitations, and future applications in acute-care
settings.THE LANCET, Infectious Diseases,Vol-4,June2004
Pusterla N, Magigan JE, Leutenegger CM(2006)Real time
polymerase chain reaction:Novel molecular diagnostic tool
for equine infectious diseases.J Vet Intern Med2006;20:3-12.
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THANK YOU !!!