Molecular Methods Molecular Methods in Diagnosis of in Diagnosis of

Infectious DiseasesInfectious Diseases

Dr.T.V.Rao MDDr.T.V.Rao MD

Diagnostic methods in Diagnostic methods in MMicrobiologyicrobiology

Task of the method – to make the microorganism “visible” and

“measureable”

Cultivation Bio-testing Immunological methods Biochemical methods Microscopy Molecular methods

Watson and Crick’s Discovery Watson and Crick’s Discovery of DNA -Path to Molecular of DNA -Path to Molecular

MedicineMedicine

Traditional Microbial Traditional Microbial Diagnostic MethodsDiagnostic Methods

Morphology Morphology Staining propertiesStaining properties Grow on different Grow on different

laboratory condtions laboratory condtions and media.and media.

Phenotyping Phenotyping protocols use purely protocols use purely biological biological phenomena and phenomena and usually refer to usually refer to study of Proteinsstudy of Proteins

Disadvantages of Disadvantages of Phenotyping MethodsPhenotyping Methods

Lack of ReproducibilityLack of Reproducibility Poor Discriminatory powerPoor Discriminatory power Difficulties in Typing Difficulties in Typing

Genotyping MethodsGenotyping Methods Genetic methods Genetic methods

generally seek to generally seek to detect detect polymorphisim at polymorphisim at the level of nucleic the level of nucleic acidacid

Genotypes are more Genotypes are more specific, more easily specific, more easily quantified and quantified and standardized among standardized among the different the different organismsorganisms

Basic requirementBasic requirement

The genome is The genome is unique in each unique in each individualindividual

Ultimate Ultimate discriminatory step discriminatory step would be to would be to sequence the entire sequence the entire genome of every genome of every organism, but not organism, but not practical or practical or economicaleconomical

What is PracticableWhat is Practicable

Several methods in Several methods in detecting nuclei detecting nuclei acid polymorphisim acid polymorphisim in a chosen genetic in a chosen genetic marker are marker are commonly used to commonly used to target the genome target the genome or organism.or organism.

Complete genomic Complete genomic configurationconfiguration

Restriction EndonuleasesRestriction Endonuleases It was discovered that a It was discovered that a

type of bacterial type of bacterial enzyme was found to enzyme was found to have the ability to cut have the ability to cut DNA in a test tube. DNA in a test tube. These restriction These restriction endonulease, so named endonulease, so named because they cut because they cut double stranded DNA at double stranded DNA at restricted sites, were restricted sites, were discovered as a natural discovered as a natural part of the bacterial part of the bacterial machinery. machinery.

Restriction endonulease EcoRIRestriction endonulease EcoRI cuts cuts double-stranded DNA and generates sticky double-stranded DNA and generates sticky

endsends. . 

What is the Choice of What is the Choice of MarkerMarker

Genetic markers Genetic markers are nucleotide are nucleotide sequences within sequences within the genome that the genome that are polymorphic are polymorphic (with differences) (with differences) between strains of between strains of the same the same organisms organisms

Choice of MarkersChoice of Markers

Markers depend on Markers depend on Type of study,Type of study,

Recent outbreakRecent outbreak

Rapid evolving Rapid evolving markers.markers.

Markers related to Markers related to evolution of evolution of organism over organism over years require more years require more stable markersstable markers

Kary Mullis - Nobel prize in Kary Mullis - Nobel prize in 19931993

Kary Mullis shared Kary Mullis shared the 1993 Nobel the 1993 Nobel Prize in Chemistry Prize in Chemistry with Michael Smith. with Michael Smith. Mullis received the Mullis received the prize for his prize for his development of the development of the Polymerase Chain Polymerase Chain Reaction (PCR)Reaction (PCR)

PCR a Revolution in PCR a Revolution in ScienceScience

A process first A process first described by Kjell described by Kjell Kleppe and 1968 Nobel Kleppe and 1968 Nobel laureate H. Gobind laureate H. Gobind Khorana that allows the Khorana that allows the amplification of specific amplification of specific DNA sequences. The DNA sequences. The improvements provided improvements provided by Mullis have made by Mullis have made PCR a central technique PCR a central technique in biochemistry and in biochemistry and molecular biologymolecular biology

Polymerase Chain Polymerase Chain ReactionReaction

Polymerase chain reactionPolymerase chain reaction ( (PCRPCR) is a ) is a technique to amplify a single or few technique to amplify a single or few copies of a piece of DNA across several copies of a piece of DNA across several orders of magnitude, generating orders of magnitude, generating millions or more copies of a particular millions or more copies of a particular DNA sequence. The method relies on DNA sequence. The method relies on thermal cycling, consisting of cycles of thermal cycling, consisting of cycles of repeated heating and cooling of the repeated heating and cooling of the reaction for DNA melting and reaction for DNA melting and enzymatic replication of the DNA. enzymatic replication of the DNA.

Taq polymerase - backbone Taq polymerase - backbone of PCR technologyof PCR technology

Almost all PCR Almost all PCR applications employ applications employ a heat-stable DNA a heat-stable DNA polymerase, such polymerase, such as as Taq Taq polymerasepolymerase, an , an enzyme originally enzyme originally isolated from the isolated from the bacterium bacterium Thermus Thermus aquaticusaquaticus..

Oligonucleotide – DNA Oligonucleotide – DNA PrimersPrimers

This DNA polymerase This DNA polymerase enzymatically enzymatically assembles a new DNA assembles a new DNA strand from DNA strand from DNA building blocks, the building blocks, the nucleotides, by using nucleotides, by using single-stranded DNA as single-stranded DNA as a template and DNA a template and DNA Oligonucleotide (also Oligonucleotide (also called DNA primers), called DNA primers), which are required for which are required for initiation of DNA initiation of DNA synthesis. synthesis.

Amplified sequences are Amplified sequences are BlottedBlotted

PCR in Clinical PCR in Clinical MicrobiologyMicrobiology

Molecular detection Molecular detection has mostly come to has mostly come to the the clinical clinical microbiologymicrobiology laboratory in the laboratory in the form of PCR form of PCR technology, initially technology, initially involving single involving single round or nested round or nested procedures with procedures with detection by gel detection by gel electrophoresis. electrophoresis.

Helps Rapid DetectionHelps Rapid Detection

Polymerase chain Polymerase chain reaction (PCR) reaction (PCR) techniques have led techniques have led the way into this new the way into this new era by allowing rapid era by allowing rapid detection of detection of microorganisms that microorganisms that were previously were previously difficult or impossible difficult or impossible to detect by traditional to detect by traditional microbiological microbiological methods. methods.

Automation and Multiplex Automation and Multiplex PCRPCR

With the advent of With the advent of multiplex PCR, real-multiplex PCR, real-time PCR and time PCR and improvements in improvements in efficiency through efficiency through automation, the automation, the costs of molecular costs of molecular methods are methods are decreasing such decreasing such that the role of that the role of molecular methods molecular methods will further increase. will further increase.

Progress in Molecular Progress in Molecular MethodsMethods

Molecular methods Molecular methods have now have now progressed beyond progressed beyond identification to identification to detect antimicrobial detect antimicrobial resistance genes resistance genes and provide public and provide public health information health information such as strain such as strain characterisation by characterisation by genotyping.genotyping.

Molecular based Tests Molecular based Tests needneed

Nucleic acid-based tests used in Nucleic acid-based tests used in diagnosing infectious diseases use diagnosing infectious diseases use standard methods for isolating standard methods for isolating nucleic acids from organisms and nucleic acids from organisms and clinical material and restriction clinical material and restriction endonulease enzymes, gel endonulease enzymes, gel electrophoresis, and nucleic acid electrophoresis, and nucleic acid hybridization techniques to analyze hybridization techniques to analyze DNA or RNA DNA or RNA

Molecular diagnostics is a set of methods to study primary structure (sequence) of DNA

•Hybridization with complementary sequences

•Amplification (synthesis) of species specific sequences

PCR – polymerase chain reaction

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Advances on PCR Advances on PCR MethodsMethods

Fairly recently, a Fairly recently, a new method of PCR new method of PCR quantification has quantification has been invented. This been invented. This is called “is called “real-time real-time PCR”PCR” because it because it allows the scientist allows the scientist to actually view the to actually view the increase in the increase in the amount of DNA as it amount of DNA as it is amplified. is amplified.

REAL TIME ASSAYSREAL TIME ASSAYS

New Technologies – Real New Technologies – Real Time AssaysTime Assays

The Real Time assays are proving to better The Real Time assays are proving to better technologies technologies

1 Rapid1 Rapid 2 Quantitative measurement2 Quantitative measurement 3 Lower contamination rate3 Lower contamination rate 4 Higher sensitivity4 Higher sensitivity 5 Higher specificity5 Higher specificity 6 Easy standardization6 Easy standardization Now a new gold standard for rapid Now a new gold standard for rapid

diagnosis of virus infection in the acute diagnosis of virus infection in the acute phase samples.phase samples.

RT - PCRRT - PCR Proving to beProving to be AccurateAccurate PrecisePrecise Easy to performEasy to perform RT PCR technologies RT PCR technologies

are easy to transfer are easy to transfer research Laboratory research Laboratory protocols to protocols to Diagnostic Diagnostic LaboratoriesLaboratories

OVERVIEW of RT - PCROVERVIEW of RT - PCR

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

Real Time ReportersReal Time Reporters

All real time PCR systems rely upon All real time PCR systems rely upon the detection and quantization of the detection and quantization of fluorescent reporter, the signal of fluorescent reporter, the signal of which increases in direct proportion which increases in direct proportion of the amount of PCR product in a of the amount of PCR product in a reaction.reaction.

REAL TIME PCRREAL TIME PCRCyber GreenCyber Green

USING USING SYBER® SYBER® GREENGREEN

The simplest and The simplest and economical format economical format the reporter is the the reporter is the double strand DNA double strand DNA specific dye SYBR specific dye SYBR ® Green ® Green

Called as Molecular Called as Molecular Probe.Probe.

How SYBR Green worksHow SYBR Green works

SYBR green binds SYBR green binds to double stranded to double stranded DNA and upon DNA and upon excitation emits excitation emits light light

Thus as PCR Thus as PCR product product accumulates the accumulates the fluoresce increasesfluoresce increases

LimitationsLimitations of of SYBER®GreenSYBER®Green

AdvantagesAdvantages

InexpensiveInexpensive

Easy to UseEasy to Use

Sensitive Sensitive

DisadvantagesDisadvantages

SYBR green will bind SYBR green will bind to any double to any double stranded DNA in a stranded DNA in a reaction, may result reaction, may result in an overestimation in an overestimation of the target of the target concentrationconcentration

Other Emerging Other Emerging AlternativesAlternatives

Two most popular Two most popular alternatives to SYBR alternatives to SYBR green are green are TaqMan®TaqMan® and and Molecular Molecular BeaconsBeacons..

Both technologies Both technologies depend on depend on hybridization probes hybridization probes relying on fluorescence relying on fluorescence resonance energy resonance energy transfer.( FRET) and transfer.( FRET) and quantizationquantization

TaqMANTaqMAN®®

TaqMANTaqMAN® Sequencing® Sequencing

TaqMANTaqMAN®® probesprobes

Documentation of Documentation of AmplificationAmplification

The light emitted from The light emitted from the dye in the excited the dye in the excited state is received by a state is received by a computer and shown computer and shown on a graph display, on a graph display, such as this, showing such as this, showing PCR cycles on the X-PCR cycles on the X-axis and a logarithmic axis and a logarithmic indication of intensity indication of intensity on the Y-axis. on the Y-axis.

Molecular Beacons

Molecular Beacons Uses FRET

Fluorescence Resonance Energy Transfer

Uses two sequence specific Oligonucleotide labelled with

fluorescent dyes

Molecular Beacons – RT Molecular Beacons – RT PCRPCR

Molecular beacons are Molecular beacons are designed to adopt a designed to adopt a hairpin structure while hairpin structure while free in solution, free in solution, brining the fluorescent brining the fluorescent dye and quencher in dye and quencher in close proximity. When close proximity. When a molecular beacon a molecular beacon hybridizes to a target hybridizes to a target the fluorescent dye the fluorescent dye emits light upon emits light upon irradiation, and rebind irradiation, and rebind to target in every to target in every cycle for signal cycle for signal measurement.measurement.

Loop Mediated Isothermal Loop Mediated Isothermal Amplification (LAMP)Amplification (LAMP)

Loop mediated isothermal Loop mediated isothermal amplification is a simple, rapid, amplification is a simple, rapid, specific and cost effective nucleic specific and cost effective nucleic acid amplification method acid amplification method characterized by use of 8 distinct characterized by use of 8 distinct regions on the target gene.regions on the target gene.

The amplification proceeds at a The amplification proceeds at a constant temperature using strand constant temperature using strand displacement reaction.displacement reaction.

LAMPLAMP

Amplification and Amplification and detection of gene can detection of gene can be completed in a be completed in a single step, by single step, by incubating the mixture incubating the mixture of samples, primers of samples, primers DNA polymerase with DNA polymerase with strand displacement strand displacement activity and substrates activity and substrates at a constant at a constant temperature of 63temperature of 6300c.c.

LAMPLAMP Compared with PCR, Compared with PCR,

and real time PCR, the and real time PCR, the LAMP has advantages LAMP has advantages of reaction simplicity of reaction simplicity and detection and detection sensitivity.sensitivity.

The higher sensitivity The higher sensitivity and specificity of LAMP and specificity of LAMP reaction is attributed to reaction is attributed to continuous continuous amplification under amplification under isothermal condition isothermal condition employing six primers employing six primers recognizing eight recognizing eight distinct regions of the distinct regions of the target.target.

Advantages of LAMPAdvantages of LAMP

LAMP functions on isothermal LAMP functions on isothermal amplification.amplification.

LAMP does not require any thermal LAMP does not require any thermal cycler and thus cane be performed cycler and thus cane be performed even with water bath/heating blockeven with water bath/heating block

LAMP method do not require LAMP method do not require sophisticated temperature control sophisticated temperature control devicesdevices

Cost effectiveCost effective

Lesser False Positives in Lesser False Positives in LAMPLAMP

In LAMP both In LAMP both amplification and amplification and detection occur detection occur simultaneously during simultaneously during the exponential phase the exponential phase without going through without going through the plateau phase the plateau phase where the non where the non spurious amplification spurious amplification leads to lower leads to lower sensitivity and false sensitivity and false positivity.positivity.

Loop Mediated Isothermal Loop Mediated Isothermal Amplification in Clinical DiagnosisAmplification in Clinical Diagnosis

LAMP technology proving to be ideal in LAMP technology proving to be ideal in detection of DNA or RNA of the pathogenic detection of DNA or RNA of the pathogenic organisms organisms

Proving to be highly efficient in diagnosis Proving to be highly efficient in diagnosis of Viral and Bacterial infections,of Viral and Bacterial infections,

LAMP is capable of detecting the presence LAMP is capable of detecting the presence of pathogenic agents earlier than PCRof pathogenic agents earlier than PCR

LAMP proving an efficient LAMP proving an efficient TechnologyTechnology

A one step single tube A one step single tube real time accelerated real time accelerated reverse transcription reverse transcription loop mediated loop mediated isothermal amplification isothermal amplification (RT-LAMP(RT-LAMP) assays for ) assays for rapid detection of some rapid detection of some recently emerged viral recently emerged viral pathogen eg West Nile, pathogen eg West Nile, SARS, Dengue, SARS, Dengue, Japanese encephalitis Japanese encephalitis Chikungunya Norwalk, Chikungunya Norwalk, H5N1 highly pathogenic H5N1 highly pathogenic avian influenza., and avian influenza., and CMV,HPV,VZVCMV,HPV,VZV

Multiplex PCRMultiplex PCR TaqMan probes and TaqMan probes and

Molecular beacons Molecular beacons allow multiple DNA allow multiple DNA species to be species to be measured in the measured in the same sample same sample ( Multiplex PCR) ( Multiplex PCR) since fluorescent since fluorescent dyes with different dyes with different emission spectra emission spectra may be attached to may be attached to different probesdifferent probes

Uses of Automated RT - Uses of Automated RT - PCRPCR

Several viral infections can be detected in Several viral infections can be detected in acute phase serum samples.acute phase serum samples.

Increasing used in for early and accurate Increasing used in for early and accurate detection of all most human viruses includingdetection of all most human viruses including

Measles, Mumps, Herpes simplex Measles, Mumps, Herpes simplex viruses, Rota viruses Noro viruses, Rota viruses Noro virus,Influenzae virus type A and B, virus,Influenzae virus type A and B, Respiratory Syncitical virus, SARS, Respiratory Syncitical virus, SARS, Dengue Japanese Encephalitis, Hepatitis Dengue Japanese Encephalitis, Hepatitis B and C, West Nile, Chikungunya,HIV, B and C, West Nile, Chikungunya,HIV, Avian flu virus,Avian flu virus,

Multiplex PCR in Real Multiplex PCR in Real TimeTime

Multiplex real time Multiplex real time quantitative RT-quantitative RT-PCR assays have PCR assays have been developed for been developed for simultaneous simultaneous detection detection identification and identification and quantification of quantification of HBV, HCV and HIV-! HBV, HCV and HIV-! In plasma and In plasma and Serum samples.Serum samples.

Real-Time PCR MethodReal-Time PCR MethodMolecular BeaconsMolecular Beacons

Molecular beacons Molecular beacons are short segments are short segments of single-stranded of single-stranded DNA (Figure 1). The DNA (Figure 1). The sequence of each sequence of each molecular beacon molecular beacon must be must be customized to customized to detect the PCR detect the PCR product of interest. product of interest.

Molecular methods are necessary if Molecular methods are necessary if the traditional methods provide poor the traditional methods provide poor

resultsresults

Microscopy gives false positive results - Microscopy gives false positive results -

- - T.vaginalis, T.vaginalis, N.gonorrhoeaeN.gonorrhoeae

Intracellular pathogens – Intracellular pathogens – viruses, M.genitalium viruses, M.genitalium Low sensitivityLow sensitivity – – Chlamydia Chlamydia

sp.,Neisseria sp.sp.,Neisseria sp. Seropositivity is common – Seropositivity is common – Chlamydia spChlamydia sp.. Subtyping is mandatory – Subtyping is mandatory – HSV, HPV, HCVHSV, HPV, HCV Microbial growth is slow – Microbial growth is slow – M. tuberculosisM. tuberculosis

How are you going to How are you going to measure the PCR measure the PCR

productproduct DirectlyDirectly

– Sybr green Sybr green – Quality of primers criticalQuality of primers critical

IndirectlyIndirectly– In addition to primers, add a In addition to primers, add a

fluorescently labeled hybridization probefluorescently labeled hybridization probe– Many different approaches to this, see Many different approaches to this, see

Bustin Bustin J.Mol.Endocrinol. (2000) 25:169

Importance of controlsImportance of controls

Negative control (no DNA)Negative control (no DNA)– checks reagents for contaminationchecks reagents for contamination

No reverse transcriptase controlNo reverse transcriptase control– detects if signal from contaminating detects if signal from contaminating

DNADNA Positive controlPositive control

– checks that reagents and primers workchecks that reagents and primers work– especially importance if trying to show especially importance if trying to show

absence of expression of a geneabsence of expression of a gene

StandardsStandards

Same copy number in all cellsSame copy number in all cells Expressed in all cellsExpressed in all cells Medium copy number advantageous Medium copy number advantageous

– correction more accuratecorrection more accurate Reasonably large intronsReasonably large introns No pseudo geneNo pseudo gene No alternate splicing in region you No alternate splicing in region you

want to PCRwant to PCR

Real-time PCR Real-time PCR applicationsapplications

Quantitation of gene expressionQuantitation of gene expression Pathogen detectionPathogen detection Viral quantitationViral quantitation Array verificationArray verification Drug therapy efficacyDrug therapy efficacy DNA damage measurementDNA damage measurement Quality control and assay validationQuality control and assay validation GenotypingGenotyping

Establishing PCR laboratory

Sample handling

DNA preparation

Clean room

Stock solutions

Laboratory

Mixing site

Thermocycler

Amplification

Detection

Documentation

QC & QAQuality control & assurance

R & D(Research and development)

Alternatives: - commercial kits - robots + kits

No alternative

Emerging Emerging Technologies in Technologies in

Molecular DiagnosisMolecular Diagnosis

QIAGEN One Step RT-PCR QIAGEN One Step RT-PCR KitKit

The QIAGEN One Step The QIAGEN One Step RT-PCR Kit is designed RT-PCR Kit is designed for easy and sensitive for easy and sensitive one-step RT-PCR using one-step RT-PCR using any RNA template. A any RNA template. A unique enzyme unique enzyme combination and combination and specially developed specially developed reaction buffer ensure reaction buffer ensure efficient reverse efficient reverse transcription and PCR transcription and PCR in one tube. in one tube.

RT-PCR in one step RT-PCR in one step The Robus™ T I Kit is baseThe Robus™ T I Kit is base

RobusT RT-PCR RobusT RT-PCR Kits perform Kits perform cDNA synthesis cDNA synthesis and PCR and PCR amplification of amplification of cDNA cDNA successively in a successively in a single tube single tube during a during a continuous continuous thermal cyclingthermal cycling

Uses and Advantages in Uses and Advantages in Testing by PCR MethodsTesting by PCR Methods

Clinical diagnostics: detection and Clinical diagnostics: detection and quantification of infectious microorganisms, quantification of infectious microorganisms, cancer cells and genetic disorders cancer cells and genetic disorders

Capable of amplifying long targets, up to Capable of amplifying long targets, up to 6.0 kb 6.0 kb

One-tube system allows rapid, sensitive and One-tube system allows rapid, sensitive and reproducible analysis of RNA with minimal reproducible analysis of RNA with minimal risk of sample contamination risk of sample contamination

Amplifies products from a wide variety of Amplifies products from a wide variety of total RNA or mRNA sources total RNA or mRNA sources

AdvantagesMolecular methods

•High sensitivity and specificity•Detects pathogen, not immune response•Quick results•High transport toleration

In-house (home-brew) PCR methods•Cost effective•High sensitivity•High quality•Fast implementation of scientific discoveries•Customer friendly

R&D is absolutely necessary

ESTABLISHMENT OF A PCR LABORATORY

To perform PCR for the repetitive detection of a specific sequence, three distinct laboratory areas are required. The specific technical operations, reagents ,and personnel considerations

Prevention of Prevention of Contamination in PCR Contamination in PCR

LaboratoryLaboratory PCR contamination be considered as

a form of infection. If standard sterile techniques that would be applied to tissue culture or microbiological manipulations are applied to PCR, then the risk of contamination will be greatly reduced. Above all else, common sense should prevail.

Avoiding contaminationAvoiding contamination The single most important source of PCR

product contamination is the generation of aerosols of PCR amplicons that is associated with the post-PCR analysis. Methods for eliminating this aerosol range from physical design of laboratories and use of specific pipettes to chemical and enzymatic approaches. The choice of method is often dependent on the frequency of amplification of a target amplicon and the relative amounts and concentrations of the amplicons created by the PCR.

Created for Awareness Created for Awareness on Emerging on Emerging

Technologies in Technologies in Developing WorldDeveloping World

Dr.T.V.Rao MDDr.T.V.Rao MD

EmailEmail

doctortvrao@gmail.comdoctortvrao@gmail.com