pcr in diagnostic medicne

8/4/2019 PCR in diagnostic medicne

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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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pcr in diagnostic medicne

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