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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
Genotypic AssaysGenotypic Assays
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
Genotypic AssaysGenotypic Assays
– 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
Direct Detection of Virus or Viral Direct Detection of Virus or Viral Antigen: Electron MicroscopyAntigen: Electron Microscopy
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.
Direct Detection of Virus or Viral Direct Detection of Virus or Viral Antigen: Electron MicroscopyAntigen: Electron Microscopy
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
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
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
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 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
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
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.