Viral Diagnostics Jonathan Gubbay Ontario Agency for Health Protection and Promotion Public Health Laboratory-Toronto

Viral DiagnosticsViral Diagnostics

Jonathan GubbayJonathan Gubbay

Ontario Agency for Health Protection and PromotionOntario Agency for Health Protection and PromotionPublic Health Laboratory-TorontoPublic Health Laboratory-Toronto

OverviewOverview

Clinical virology lab can provide significant Clinical virology lab can provide significant benefit to patient carebenefit to patient care

Traditionally epidemiologic and academic Traditionally epidemiologic and academic rolerole

Current rapid assays impact on Current rapid assays impact on therapeutic and public health decisions. therapeutic and public health decisions. – Change largely due to molecular methodsChange largely due to molecular methods

Impact of PCR on virologyImpact of PCR on virology

Recent identification of several respiratory Recent identification of several respiratory virusesviruses– MetapneumovirusMetapneumovirus– Multiple coronaviruses: SARS, 229E, NL63, Multiple coronaviruses: SARS, 229E, NL63,

OC43, HKU1. OC43, HKU1. – Human bocavirusHuman bocavirus– Polyomaviruses KI, WUPolyomaviruses KI, WU

Why Expanding Role for Why Expanding Role for Diagnostic Virology LabDiagnostic Virology Lab

Increased pool of immunocompromisedIncreased pool of immunocompromised

Increasing antiviral agents Increasing antiviral agents

Results in increasing demand for rapid Results in increasing demand for rapid methods, viral load testing, antiviral methods, viral load testing, antiviral susceptibility, genotyping. susceptibility, genotyping.

Methods in use in virology.Methods in use in virology.

Detecting Active Infection:Detecting Active Infection:– Electron MicroscopyElectron Microscopy– Viral culture Viral culture – Detection of viral antigens Detection of viral antigens – Detection of viral nucleic acid.Detection of viral nucleic acid.– HistopathologyHistopathology

Assessing virus-specific immune responseAssessing virus-specific immune response– Serologic testing (won’t cover today)Serologic testing (won’t cover today)

Specimen choice and collectionSpecimen choice and collection

Specimen quality limits test qualitySpecimen quality limits test quality

Pathogen detection depends on:Pathogen detection depends on:– Appropriate collection site.Appropriate collection site.– Proper timing of specimen collection.Proper timing of specimen collection.– Effective and timely processing of sufficient Effective and timely processing of sufficient

specimen.specimen.

Specimen storage and transportSpecimen storage and transport

Keep specimens other than blood at 4ºCKeep specimens other than blood at 4ºC

If delay >24hrs, freeze at 70ºC or below.If delay >24hrs, freeze at 70ºC or below.

Avoid any storage at -20ºC: greater loss in Avoid any storage at -20ºC: greater loss in infectivityinfectivity

Nonenveloped viruses (adenovirus, Nonenveloped viruses (adenovirus, enteroviruses) more stable than enveloped enteroviruses) more stable than enveloped (e.g. RSV, VZV, CMV). (e.g. RSV, VZV, CMV).

Viral Transport MediumViral Transport Medium

Salt solution – ensures proper ionic Salt solution – ensures proper ionic concentrationsconcentrations

Buffer - maintains pHBuffer - maintains pH

Protein - for virus stabilityProtein - for virus stability

Antibiotics or antifungals – to prevent Antibiotics or antifungals – to prevent contaminationcontamination

Cell CultureCell Culture

Viruses are obligate intracellular organisms – Viruses are obligate intracellular organisms – require living cells for virus isolationrequire living cells for virus isolation

Advantages:Advantages:– Relatively sensitive and specificRelatively sensitive and specific– Can detect many different viruses Can detect many different viruses – Provides a viral isolate for further characterization Provides a viral isolate for further characterization

(serotyping, genotyping, susceptibility)(serotyping, genotyping, susceptibility)

Cell culture –limitationsCell culture –limitations

Certain viruses don’t grow or grow slowlyCertain viruses don’t grow or grow slowly

Other techniques for detecting viral Other techniques for detecting viral infection more cost effectiveinfection more cost effective

Successful culture depends on viability of Successful culture depends on viability of virus in specimenvirus in specimen

Standard Cell CulturesStandard Cell Cultures

Originally used animals and embryonated Originally used animals and embryonated eggs. eggs.

Monolayer cell culture techniques (1933)Monolayer cell culture techniques (1933)

Roller tube cell cultures (1940)Roller tube cell cultures (1940)

Standard Cell CulturesStandard Cell Cultures

Primary cellsPrimary cells– 1-2 passages1-2 passages

Diploid (semicontinuous) cellsDiploid (semicontinuous) cells– 20-50 passages20-50 passages

Heteroploid cells.Heteroploid cells.– Indefinite passagesIndefinite passages

Cytopathic EffectCytopathic Effect

Monitor tube cultures daily initiallyMonitor tube cultures daily initially

Monitor for 10-21 daysMonitor for 10-21 days

Compare to uninoculated controls from Compare to uninoculated controls from same batchsame batch

Rounding, refractile cells, syncytium Rounding, refractile cells, syncytium formation, cell destructionformation, cell destruction

Cell culture – clues to virus Cell culture – clues to virus causing CPEcausing CPE

Type of specimenType of specimen

Cell line displaying CPECell line displaying CPE

Type of CPEType of CPE

Hemadsorbing virusesHemadsorbing viruses

Orthomyxoviruses (influenza) and some Orthomyxoviruses (influenza) and some paramyxoviruses (parainfluenza, measles, paramyxoviruses (parainfluenza, measles, mumps)mumps)

Insert viral glycoproteins (haemaglutinin)Insert viral glycoproteins (haemaglutinin)

into host cell membrane.into host cell membrane.

Promotes attachment of RBC of certain species Promotes attachment of RBC of certain species (e.g guinea pig) to cell membrane. (e.g guinea pig) to cell membrane.

Interference Interference

Rubella virus growing in monkey kidney Rubella virus growing in monkey kidney cells inhibits infection with echovirus 2. cells inhibits infection with echovirus 2.

Adenovirus CPEAdenovirus CPE

RSV - syncytiaRSV - syncytia

Cell culture – definitive Cell culture – definitive identificationidentification

Reaction with monoclonal antibodies. Reaction with monoclonal antibodies.

Antibodies chemically conjugated to a Antibodies chemically conjugated to a fluorochrome. fluorochrome.

Indirect or direct immunofluorescence. Indirect or direct immunofluorescence.

Neutralization – use monospecific or Neutralization – use monospecific or pooled antisera to prevent infection of pooled antisera to prevent infection of susceptible cells (enterovirus serotyping)susceptible cells (enterovirus serotyping)

Shell Vial CultureShell Vial Culture

System which detects viral infection prior to CPE System which detects viral infection prior to CPE developing.developing.

Low speed centrifugation enhances infection.Low speed centrifugation enhances infection.

First used for CMVFirst used for CMV– MRC5 cells, stain for early antigen protein after 18-MRC5 cells, stain for early antigen protein after 18-

48hr incubation48hr incubation

Also developed for other viruses: VZV, HSV, Also developed for other viruses: VZV, HSV, adenovirus, respiratory virusesadenovirus, respiratory viruses

CMV early antigen stainingCMV early antigen staining

Mixed Cell CultureMixed Cell Culture

Shell vial culture with mixed cell linesShell vial culture with mixed cell lines

Allows detection of wider range of viruses.Allows detection of wider range of viruses.

R-MixR-MixTMTM: mink lung + A549: mink lung + A549– RSV, parainfluenza 1-3, influenza, RSV, parainfluenza 1-3, influenza,

adenovirusesadenoviruses

R-Mix Too: MDCK + A549R-Mix Too: MDCK + A549– Above plus metapneumovirusAbove plus metapneumovirus

E-Mix: BGMK + A549E-Mix: BGMK + A549

Recent R-Mix Too vs Rhesus Recent R-Mix Too vs Rhesus Monkey Kidney at PHL-TorontoMonkey Kidney at PHL-Toronto

257 stored primary samples; 194 positive257 stored primary samples; 194 positiveR-Mix shell vials stained at 1,2 and 5dR-Mix shell vials stained at 1,2 and 5dTube culture monitored for 10dTube culture monitored for 10dR-Mix detected 67.5% of and tube culture 45.9% R-Mix detected 67.5% of and tube culture 45.9% of previously positivesof previously positivesOf pos R-Mix, 73.3% and 95.5% detected after 1 Of pos R-Mix, 73.3% and 95.5% detected after 1 and 2 days. and 2 days. Of pos TC, 2%, 25% and 60% isolated by day Of pos TC, 2%, 25% and 60% isolated by day 1,2 and 7; 36% took 10 days to be isolated.1,2 and 7; 36% took 10 days to be isolated.

Genetically Engineered Cell LinesGenetically Engineered Cell Lines

HSVHSV– Baby hamster kidney (BHK-21) transformed with an Baby hamster kidney (BHK-21) transformed with an

HSV-inducible promoter (UL39 gene)HSV-inducible promoter (UL39 gene)– UL39 attached to functional E Coli UL39 attached to functional E Coli ββ-galactosidase -galactosidase

gene.gene.– ββ-galactosidase activity induced by HSV 1 or 2 -galactosidase activity induced by HSV 1 or 2

infection. infection. – Addition of substrate (X-Gal) for this enzyme results in Addition of substrate (X-Gal) for this enzyme results in

coloured product in HSV-infected cells. coloured product in HSV-infected cells. – Commercially available as enzyme-linked virus-Commercially available as enzyme-linked virus-

inducible system (ELVIS HSV ID). inducible system (ELVIS HSV ID).

Genetically Engineered Cell LinesGenetically Engineered Cell Lines

Rapid detection after overnight incubation. Rapid detection after overnight incubation.

Antiviral Susceptibility TestingAntiviral Susceptibility Testing

May do for herpesviruses when:May do for herpesviruses when:– HSV or VZV cutaneous lesions fail to resolve HSV or VZV cutaneous lesions fail to resolve

or appearance of new ones while on oral or appearance of new ones while on oral therapy.therapy.

– Progressive retinal or visceral CMV disease Progressive retinal or visceral CMV disease while patient on therapy.while patient on therapy.

Ganciclovir-resistant CMVGanciclovir-resistant CMV

First reported ganciclovir R CMV among First reported ganciclovir R CMV among HIV-infected with CD4 <50 x 10HIV-infected with CD4 <50 x 1099//

Emerging problem in HSCT and SOT Emerging problem in HSCT and SOT patients. patients.

R testing now recommended for HIV R testing now recommended for HIV treatment failure; many do routinely at treatment failure; many do routinely at diagnosisdiagnosis

CDC Issues Interim Recommendations for the Use of Influenza Antiviral Medications in the Setting of Oseltamivir Resistance among Circulating Influenza A (H1N1) Viruses, 2008-09 Influenza Season

Friday, December 19, 2008, 11:50 EST (11:50 AM EST)

http://www2a.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?AlertNum=00279

Resistance testing now important

in influenza therapy

Phenotypic assaysPhenotypic assays

Measure effect of antiviral drug on growth Measure effect of antiviral drug on growth of a virus. of a virus. Measured by infectivity (plaque reduction), Measured by infectivity (plaque reduction), viral antigen or viral nucleic acid viral antigen or viral nucleic acid production, enzyme activity. production, enzyme activity. Directly measure and quantify effects of Directly measure and quantify effects of antivirals on growth. antivirals on growth. Slow, labour intensive, difficult to Slow, labour intensive, difficult to standardise standardise

Phenotypic assaysPhenotypic assays

Expressed as drug concentration that Expressed as drug concentration that inhibits 50% or 90% of viral growth (ICinhibits 50% or 90% of viral growth (IC50 50

and ICand IC9090) relative to a no drug control.) relative to a no drug control.

Phenotypic susceptibility assays in use for Phenotypic susceptibility assays in use for herpesviruses (CMV, HSV, VZV), herpesviruses (CMV, HSV, VZV), influenza and HIV-1. influenza and HIV-1. Plaque reduction assay for HSV Plaque reduction assay for HSV susceptibility approved by CLSI. susceptibility approved by CLSI.

Plaque Reduction AssayPlaque Reduction Assay

Proposed breakpoints for HSV and CMV Proposed breakpoints for HSV and CMV S, I, R based on ICS, I, R based on IC5050. .

Many variables effect resultsMany variables effect results– Cell lineCell line– Viral inoculumViral inoculum– Incubation periodIncubation period

Plaque Reduction AssayPlaque Reduction Assay

http://pathmicro.med.sc.edu/mhunt/plaque.jpg

Phenotypic susceptibility to Phenotypic susceptibility to neuraminidase inhibitorsneuraminidase inhibitors

Directly measure NA activityDirectly measure NA activity

Viral NA incubated with different Viral NA incubated with different concentrations of NA inhibitors.concentrations of NA inhibitors.

Fluorescent or chemiluminescent Fluorescent or chemiluminescent substrate added and quantitated. substrate added and quantitated.

Phenotypic assays for HIV Phenotypic assays for HIV susceptibilitysusceptibility

Traditionally measured p-24 antigen in cell Traditionally measured p-24 antigen in cell culture by EIA in presence of antiviral culture by EIA in presence of antiviral drug.drug.Recombinant virus phenotypic assays Recombinant virus phenotypic assays – Insertion of RT and polymerase from patient Insertion of RT and polymerase from patient

into a vector consisting of a rapidly replicating into a vector consisting of a rapidly replicating viral strain and a reporter gene (luciferase). viral strain and a reporter gene (luciferase).

– Measure viral growth in presence of drug Measure viral growth in presence of drug compared to wild type virus. (Phenosense, compared to wild type virus. (Phenosense, Antivirogram)Antivirogram)

Genotypic AssaysGenotypic Assays

Allow rapid detection of genetic mutations Allow rapid detection of genetic mutations associated with antiviral drug resistance. associated with antiviral drug resistance.

1. Nucleic acid amplification1. Nucleic acid amplification

2. Sequencing of amplified product2. Sequencing of amplified product

3. Compare amplicon sequence to reference 3. Compare amplicon sequence to reference strainstrain


Most useful when a discrete number of Most useful when a discrete number of known resistance mutationsknown resistance mutations– CMV mutations causing ganciclovir CMV mutations causing ganciclovir

resistance. resistance. UL 97 (protein kinase) – ganciclovirUL 97 (protein kinase) – ganciclovir UL54 (DNA polymerase) – ganciclovir, foscarnet, UL54 (DNA polymerase) – ganciclovir, foscarnet, cidofivircidofivir

– Antiretroviral-refractory HIV infections.Antiretroviral-refractory HIV infections.Amplification and sequencing of RT and Amplification and sequencing of RT and polymerase genespolymerase genes


– Lamivudine R in Hep BLamivudine R in Hep B– M2 mutations in amantadane R M2 mutations in amantadane R

Influenza A. Influenza A. – NA (H275Y) and/or hemagluttinin NA (H275Y) and/or hemagluttinin

mutations in Influenza A associated mutations in Influenza A associated with NA inhibitor R. with NA inhibitor R.

Direct Detection of Virus or Viral Direct Detection of Virus or Viral Antigen: Electron MicroscopyAntigen: Electron Microscopy

Superseded by other methods in most Superseded by other methods in most labslabs

Still important for rapid detection of viruses Still important for rapid detection of viruses in clinical samples.in clinical samples.

Able to detect multiple pathogensAble to detect multiple pathogens


Specimen absorbed onto thin Specimen absorbed onto thin plastic/carbon film.plastic/carbon film.

Applied to surface of EM grid before Applied to surface of EM grid before staining.staining.

Can use positive or negative stain (most Can use positive or negative stain (most common – phosphotungstic acid)common – phosphotungstic acid)– Penetrates virion and provides contrast for Penetrates virion and provides contrast for

visualization of cell surface.visualization of cell surface.


QuickQuick

Looks for many virusesLooks for many viruses

Useful if unknown pathogenUseful if unknown pathogen

Less prone to cross contamination vs molecular. Less prone to cross contamination vs molecular.

Expensive equipment, need expertise to readExpensive equipment, need expertise to read

Not well suited to screening large numbers of Not well suited to screening large numbers of samples. samples.

Low sensitivity – need 10Low sensitivity – need 1055-10-108 8 viral particles/ml viral particles/ml to detect. to detect.

AdenovirusAdenovirus

http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Safrica/adeno3.htm

Human RotavirusHuman Rotavirus

Paramyxovirus (Parainfluenza)Paramyxovirus (Parainfluenza)

Histopathology/CytologyHistopathology/Cytology

Some viruses produce characteristic Some viruses produce characteristic cytologic/histologic changescytologic/histologic changes

Not enough time to go into detail now….Not enough time to go into detail now….

Direct Detection of Viruses: Direct Detection of Viruses: ImmunoassaysImmunoassays

Utilize antibodies (monoclonal or Utilize antibodies (monoclonal or polyclonal) against specific viral polyclonal) against specific viral antigen/antigens. antigen/antigens.

Ag/Ab complexes detected by:Ag/Ab complexes detected by:– Direct visualizationDirect visualization– Solid Phase Enzyme immunoassaysSolid Phase Enzyme immunoassays– Enzyme Linked immunoassaysEnzyme Linked immunoassays

Direct Visualization: direct methodDirect Visualization: direct method

Direct or indirect staining methodsDirect or indirect staining methods

Direct: antibody conjugated with either an Direct: antibody conjugated with either an enzyme (e.g horseradish peroxidase) or enzyme (e.g horseradish peroxidase) or fluorescent label (e.g FITC). fluorescent label (e.g FITC).

Substrated added to Ag/Ab-Conjugate on Substrated added to Ag/Ab-Conjugate on glass slide, resulting in colour. glass slide, resulting in colour.

Ag/Ab-FITC visualized using fluorescent Ag/Ab-FITC visualized using fluorescent microscope.microscope.

Direct visualization: indirect methodDirect visualization: indirect method

An unlabelled (e.g mouse) antibody is An unlabelled (e.g mouse) antibody is added to bind to the antigen of interestadded to bind to the antigen of interestA second antibody labelled with conjugate A second antibody labelled with conjugate is added.is added.Visualized using substrate as with direct Visualized using substrate as with direct methodmethodIndirect allows amplification of signal –Indirect allows amplification of signal –many more labelled antibodies can bind to many more labelled antibodies can bind to the intermediate unlabelled one. the intermediate unlabelled one.

Direct and indirect Direct and indirect immunofluorescenceimmunofluorescence

DFA – direct immunofluoresence. DFA – direct immunofluoresence.

IFA – indirect immunofluoresence. IFA – indirect immunofluoresence.

Widely used in virologyWidely used in virology

Cheap, easy to do. Cheap, easy to do.

Monoclonal abodies give high specificity.Monoclonal abodies give high specificity.

Pooling of monoclonals can detect multiple Pooling of monoclonals can detect multiple viruses – e.g resp virus DFAviruses – e.g resp virus DFA

Used for CMV pp65 antigenemia quantificationUsed for CMV pp65 antigenemia quantification

DFA RSVDFA RSV

CMV pp65 antigenCMV pp65 antigen

http://ibmi.mf.uni-lj.si/acta-apa/acta-apa-00-3/Marin.html

ELISAs/EIAsELISAs/EIAs

Surface of solid phase (microtitre plate) Surface of solid phase (microtitre plate) coated with antibodycoated with antibody

Antigen of interest binds if present.Antigen of interest binds if present.

Second enzyme-conjugated antibody Second enzyme-conjugated antibody addedadded

Substrate added and colour Substrate added and colour generated/read by spectrophotometer. generated/read by spectrophotometer.

http://www.dshs.state.tx.us/lab/images/eia_1.jpg

ELISAs/EIAsELISAs/EIAs

Sometimes second antibody is unlabelled and Sometimes second antibody is unlabelled and use 3use 3rdrd labelled antibody. labelled antibody.

Most give qualitative result, some quantitative.Most give qualitative result, some quantitative.

Newer EIAs use microbeads as the solid phase Newer EIAs use microbeads as the solid phase to increase surface area of contact. to increase surface area of contact. – Decreases reaction time to 30min.Decreases reaction time to 30min.

Sensitive – detect viral antigens at pico to Sensitive – detect viral antigens at pico to nanomolar (10nanomolar (10-12-12 to 10 to 10-9-9mol/litre)mol/litre)

ELISAsELISAs

Can be noncompetitive “sandwich” as described Can be noncompetitive “sandwich” as described previouslypreviouslyCompetitive – Solid phase is coated with antigen Competitive – Solid phase is coated with antigen to be detected. to be detected. – Patients sample is mixed with detecting antibody, Patients sample is mixed with detecting antibody,

which is then applied to solid phase.which is then applied to solid phase.– If sample contained antigen, less antibody is free to If sample contained antigen, less antibody is free to

bind to the solid phase with prebound antigen. bind to the solid phase with prebound antigen. – Rest of test run as before. Intensity of colour Rest of test run as before. Intensity of colour

formation inversely proportional to amount of antigen formation inversely proportional to amount of antigen in sample. in sample.

Nucleic Acid DetectionNucleic Acid Detection

Short length of viral genome makes them Short length of viral genome makes them ideal candidate for nucleic-acid based ideal candidate for nucleic-acid based diagnosisdiagnosisPCRPCR– conventional PCR – agarose gel detection of conventional PCR – agarose gel detection of

productproduct– Real-time PCR- products detected using Real-time PCR- products detected using

probes or intercalating dyes within the probes or intercalating dyes within the reaction.reaction.

– Microarrays – chip or bead based.Microarrays – chip or bead based.

PCRPCR

Each cycle of PCR doubles the number of Each cycle of PCR doubles the number of copies (amplicons)copies (amplicons)

Over 1 million amplicons after 20 cycles. Over 1 million amplicons after 20 cycles.

Primers determine sensitivity and Primers determine sensitivity and specificity of PCR reactions. specificity of PCR reactions.

©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2

5’ Exonuclease Probes

©Clinical and Laboratory Standards Institute. All rights reserved.MM3-A2

Molecular BeaconsMolecular Beacons

FRET probesFRET probes

Multiplex PCRMultiplex PCR

Multiple viruses can cause same clinical Multiple viruses can cause same clinical syndrome syndrome – Respiratory infectionsRespiratory infections

Can perform multiplex PCR assays to Can perform multiplex PCR assays to detect multiple viruses in one reaction.detect multiple viruses in one reaction.

Commercial assays to detect up to 18 Commercial assays to detect up to 18 respiratory viruses in 1 test.respiratory viruses in 1 test.

Seeplex®RV/PB18 ASE Detection

Multiplex–PCR System for the detection of 13 Respiratory Viruses (Influenza A/B virus, RSV A/B, Rhinovirus, Coronavirus OC43/HKU1, coronavirus 229E/NL63, adenovirus, parainfluenza virus 1-3m bocavirus, enterovirus

5 Pneumonia causing bacteria (Mycoplasma pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila).

Development of a Respiratory Virus Panel Test for Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Detection of Twenty Human Respiratory Viruses by Use of

Multiplex PCR and a Fluid Microbead-Based AssayMultiplex PCR and a Fluid Microbead-Based Assay

J. Mahony, et al.J. Mahony, et al.Department of Pathology and Molecular Medicine, McMaster University, and St. Joseph’s Department of Pathology and Molecular Medicine, McMaster University, and St. Joseph’s Healthcare, Hamilton,Healthcare, Hamilton,

Ontario, Canada,1 and TmBioscience Corporation, Toronto, Ontario, Canada2Ontario, Canada,1 and TmBioscience Corporation, Toronto, Ontario, Canada2

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 2965–2970 Vol. 45, JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 2965–2970 Vol. 45, No. 9No. 9

Detects 20 different respiratory virusesDetects 20 different respiratory viruses



Reverse transcriptaseReverse transcriptase

PCRPCR

Target specific primer extensionTarget specific primer extension– containing both a virus-specific

oligonucleotide sequence and a tag oligonucleotide that hybridizes to a complementary anti-tag oligonucleotide bound to 21 spectrofluorometrically labeled microspheres



J. Mahony, et al. J Clin Micro; Sept. 2007, p. 2965–2970 Vol. 45, No. 9J. Mahony, et al. J Clin Micro; Sept. 2007, p. 2965–2970 Vol. 45, No. 9

TSPE: Target specific primerextension



Biotin labelled TSPE product reacts with Biotin labelled TSPE product reacts with streptavadin/phycoerythrinstreptavadin/phycoerythrin

Flow cell with 2 lasersFlow cell with 2 lasers– Red laser detects specific bead (1 per test Red laser detects specific bead (1 per test

target)target)– If virus was present and TSPE occurred, If virus was present and TSPE occurred,

green laser detects presence of phycoerythrin green laser detects presence of phycoerythrin and gives positive signal.and gives positive signal.

Non-PCR-based Nucleic Acid Non-PCR-based Nucleic Acid Amplification SystemsAmplification Systems

Strand Displacement AmplificationStrand Displacement Amplification

Ligase Chain ReactionLigase Chain Reaction

Nucleic Acid Sequence-Based Nucleic Acid Sequence-Based AmplificationAmplification

Hybridization –Based AssaysHybridization –Based Assays

Quantitative NATsQuantitative NATs

SummarySummary

Viral diagnostics is a dynamic fieldViral diagnostics is a dynamic field

New applications of molecular technology New applications of molecular technology being introduced continuouslybeing introduced continuously

Increased therapeutic options for viral Increased therapeutic options for viral infections have increased the clinical infections have increased the clinical relevance of making a viral diagnosis.relevance of making a viral diagnosis.

Documents

Viral Diagnostics Jonathan Gubbay Ontario Agency for Health Protection and Promotion Public Health Laboratory-Toronto