Multiplex real-time PCR Multiplex real-time PCR in Clinical in Clinical
InfectiondiagnosticsInfectiondiagnosticsChristophe Olinger
Journée scientifique, 16.10.2008Hôpital de Kirchberg
SLBC 16.10.2008
OutlineOutline
II Basics of PCR and Multiplex PCRBasics of PCR and Multiplex PCR
IIII Applications in clinical diagnosticsApplications in clinical diagnostics
SLBC 16.10.2008
The basics of PCR: Common groundThe basics of PCR: Common groundBacteria
Parasites
VirusesHuman
SLBC 16.10.2008
The basics of PCR: DNAThe basics of PCR: DNA
All lifeforms are based on DNA All lifeforms are based on DNA (deoxyribonucleic acid)(deoxyribonucleic acid)
Some are based on RNA, (mRNA)Some are based on RNA, (mRNA) The paradigm of lifeThe paradigm of life
DNA (genome) RNA Protein
DNA
Replication
SLBC 16.10.2008
The basics of PCR: ReplicationThe basics of PCR: Replication
PCR is based on universal biological mechanism: PCR is based on universal biological mechanism: ReplicationReplication
Replication is performed by DNA polymerase Replication is performed by DNA polymerase (Taq polymerase) (DNA to DNA)(Taq polymerase) (DNA to DNA)
PCR: Polymerase chain reactionPCR: Polymerase chain reaction RNA to DNA: reverse transcriptaseRNA to DNA: reverse transcriptase
SLBC 16.10.2008
The basics of PCR: The componentsThe basics of PCR: The componentsTarget: DNA DNA and RNA Polymerase Building blocks: dNTP (A, C, T, G)
(+ buffer)(extraction)
Start and end points: primers
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The basics of PCR: The principlesThe basics of PCR: The principles1 molecule
2 molecules
4 molecules
8 molecules
Typical PCR: 40 cycles
With 1 molecule at 1st cycle, 1x1012 after 40
Exponential increase
SLBC 16.10.2008
The basics of PCR: Theory vs RealityThe basics of PCR: Theory vs Reality
Amplification curveAmplification curve
Exponential: Exact doubling of product is accumulating atevery cycle (assuming 100% reaction efficiency). Thereaction is very specific and precise. Linear (High Variability): The reaction components arebeing consumed, the reaction is slowing, and products arestarting to degrade. Plateau: The reaction has stopped, no more productsare being made and if left long enough, the PCR productswill begin to degrade.
1
2
3
1
2
3
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The basics of PCR: The machineThe basics of PCR: The machine
Extract, Prepare mixExtract, Prepare mix Cycling (Denaturing - annealing – elongation)Cycling (Denaturing - annealing – elongation)
SLBC 16.10.2008
The basics of PCR: The resultThe basics of PCR: The result
Visualization of result based on agarose gel Visualization of result based on agarose gel electrophoresiselectrophoresis
Identification based on the size of DNA fragments (multiplex possible)
Quantification possible but may be misleading
End-point analysis
SLBC 16.10.2008
Real-time PCR: the first generationReal-time PCR: the first generation Switch from end-point analysis to real-time analysisSwitch from end-point analysis to real-time analysis Double-stranded DNA binding markerDouble-stranded DNA binding marker
Sybrgreen Sybrgreen
Binds only to double-stranded DNA
Emits light when bound
SLBC 16.10.2008
Real-time PCR: the first generationReal-time PCR: the first generation Result expressed as Ct = threshold cycle = cycle Result expressed as Ct = threshold cycle = cycle
at which the fluorescence passes the thresholdat which the fluorescence passes the threshold
Baseline
Threshold Ct = 22
End-point irrelevant
SLBC 16.10.2008
Real-time PCR: the first generationReal-time PCR: the first generation Advantages of Sybrgreen: Advantages of Sybrgreen:
More reliable quantification (in theory)More reliable quantification (in theory) Gel not necessary (in theory)Gel not necessary (in theory)
DisadvantagesDisadvantages Binds to every double-stranded moleculeBinds to every double-stranded molecule
SLBC 16.10.2008
Real-time PCR: the current generationReal-time PCR: the current generation
Additional layer of specificityAdditional layer of specificity Higher sensitivityHigher sensitivity One example:One example:
5’ nuclease assay = TaqMan®5’ nuclease assay = TaqMan®
Q
R
Quencher
Reporter dye (fluorophore)
5’ 3’
R Q
Dual-labeled probe
X A fluorophore is a molecule that emits light of a certain wavelength after having abosrbed light of a specific, but different wavelength
A quencher is a molecule that accepts energy from a fluorophore in the form of light and dissipates this energy either in the form of light or heat
SLBC 16.10.2008
Real-time PCR: the TaqMan® assayReal-time PCR: the TaqMan® assay
Q
R
Quencher
Reporter dye (fluorophore)
2
3
SLBC 16.10.2008
Real-time PCR: MultiplexingReal-time PCR: MultiplexingMultiple Dyes
Various equipment with different channles/filters
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Real-time PCR: protocolReal-time PCR: protocol Same components as conventional PCRSame components as conventional PCR 2 primers / 1 probe per target, different fluorophores2 primers / 1 probe per target, different fluorophores Different reactions run in parallel in the same tubeDifferent reactions run in parallel in the same tube Maximum defined by equipment usedMaximum defined by equipment used
Current maximum:5 colors
SLBC 16.10.2008
Multiplex PCR: Limitation, spectral Multiplex PCR: Limitation, spectral overlappingoverlapping
SLBC 16.10.2008
Part IIPart II
Multiplex real-time PCR in clinical infectiondiagnosticsMultiplex real-time PCR in clinical infectiondiagnostics Advantages and pitfallsAdvantages and pitfalls Different clinical applicationsDifferent clinical applications
SLBC 16.10.2008
Hepatitis C is a blood-bourne infectious disease caused Hepatitis C is a blood-bourne infectious disease caused by the hepatitis C virus affecting the liver.by the hepatitis C virus affecting the liver.
200 million people worldwide are affected (~3%)200 million people worldwide are affected (~3%) Often asymptomatic, but once established can cause Often asymptomatic, but once established can cause
chronic infection of the liverchronic infection of the liver Quantitative PCR is important to follow the viral load Quantitative PCR is important to follow the viral load
of infected patients during treatment or to determine of infected patients during treatment or to determine chronicitychronicity
Simple duplex assay: hepatitis C Simple duplex assay: hepatitis C virusvirus
SLBC 16.10.2008
Simple duplex assay: hepatitis C Simple duplex assay: hepatitis C virusvirus
HCV is an enveloped, single-stranded RNA HCV is an enveloped, single-stranded RNA virusvirus
Worldwide, around 50% of variability has been Worldwide, around 50% of variability has been found between HCV strains (genotypes)found between HCV strains (genotypes)
Problem for PCR based diagnostic testsProblem for PCR based diagnostic tests
SLBC 16.10.2008
HCV duplex assay: SetupHCV duplex assay: Setup Duplex assay Duplex assay Internal Control (IC): BMV Internal Control (IC): BMV BMV: brome mosaic virus = plant RNA virusBMV: brome mosaic virus = plant RNA virus Added during extraction Added during extraction extraction control extraction control During PCR IC and HCV are amplifiedDuring PCR IC and HCV are amplified Checks for correct mix and sample additionChecks for correct mix and sample addition Checks for inhibition of polymerase/reverse Checks for inhibition of polymerase/reverse
transcriptasetranscriptase
SLBC 16.10.2008
HCV duplex assay: ExampleHCV duplex assay: Example
HCV (FAM)BMV (YAK)
4 HCV positive patients
SLBC 16.10.2008
HCV duplex assay: InterferenceHCV duplex assay: Interference
35.1236.20
35.6833.43
36.9830.06
36.4527.10
>4023.13
>4020.22
35.23-HCV 101
32.0337.78
31.7133.05
31.6130.12
31.5527.19
34.1823.17
> 4020.18
31.82-HCV 102
28.72>40
28.8533.12
28.6530.11
28.5027.12
28.2323.17
28.8420.18
29.01-HCV 103
25.43>40
25.43> 40
25.3529.63
25.1327.81
25.3323.35
25.2620.22
25.61-HCV 104
-36.45
-34.12
-29.89
-27.14
-23.23
-20.46
--No HCV
BMV 10-9BMV 10-8BMV 10-7BMV 10-6BMV 10-5BMV 10-4No BMV Duplex
Disadvantage of multiplex assays: one positive Disadvantage of multiplex assays: one positive target may inhibit other reactionstarget may inhibit other reactions
SLBC 16.10.2008
ACE assay: IntroductionACE assay: Introduction Detects Detects
AdenovirusAdenovirus CytomegalovirusCytomegalovirus Epstein-Barr virusEpstein-Barr virus Tested during transplantations, Tested during transplantations,
immunocompromised patientsimmunocompromised patients Also internal control, triplex Also internal control, triplex quadroplex quadroplex Quantification from blood samples using Quantification from blood samples using
standards with known quantitiesstandards with known quantities
SLBC 16.10.2008
ACE assay: Assay setupACE assay: Assay setup
SSSNE4321
SSSNC4321
SSSNA4321 SSSNP4321
96 well plate with three singleplexes 96 well plate with one triplex
Triplex vs Singleplex saves 18 times enzyme and buffers
A: Adenovirus positive controlC: Cytomegalovirus PCE: Epstein-Barr virus PCN: Begative controlS: Quantification standard with known quantities
ADECMV EBV
27 9
SLBC 16.10.2008
Quantification in Real-Time PCRQuantification in Real-Time PCR Efficiency of PCREfficiency of PCR Dilution series 10 fold CT 3.32, Dilutions 2 fold, Dilution series 10 fold CT 3.32, Dilutions 2 fold,
CT 1. Error range of PCR 1 – 2 CTCT 1. Error range of PCR 1 – 2 CT
y = -3.0x+32
y = -3.2x+38
y = -3.34x+35
R2 = 99,9 for allADE (CY5)CMV (VIC)EBV (FAM)
SLBC 16.10.2008
Important factors of a PCR assayImportant factors of a PCR assay Sensitivity (no false negatives)Sensitivity (no false negatives) Specificity (no false positives)Specificity (no false positives) ReproducabilityReproducability RobustnessRobustness Linearity, Dynamic RangeLinearity, Dynamic Range
Dynamic range
SLBC 16.10.2008
ACE: Example (1)
3.233.58104.354.13108.908.6194.984.9294.394.0295.765.7983.763.9984.103.9385.545.5973.172.9173.25n/a7n/a4.0163.243.3965.295.4566.656.5753.743.7953.463.6353.823.9943.883.8845.445.4046.546.4933.163.3334.133.9537.276.9323.293.4025.465.6127.247.2612.703.2514.724.581
triplex10x
single 10x
Adeno Sample
triplex10x
single 10x
CMV Sample
triplex10x
single 10x
EBV Sample
Quantities in copies/ml of 10 known positive samples tested with singleplex and triplex assays
~4% difference ~6% difference ~2% difference
SLBC 16.10.2008
ACE: Example (2)Ct values of 2 immunocompromised patients with multiple infections
11.2112.1229.2831.5029.2830.422
11.3911.4629.2728.8629.9631.301
Adeno triplex
Adeno single
CMV triplex
CMV single
EBV triplex
EBVsinglePatient
SLBC 16.10.2008
Sexually transmitted diseasesSexually transmitted diseases UrethritisUrethritis Genital swabs or urine:Genital swabs or urine:
Chlamydia trachomatis (most common, also infects Chlamydia trachomatis (most common, also infects eye)eye)
Mycoplasma genitaliumMycoplasma genitalium Neisseria gonorrhoeaeNeisseria gonorrhoeae
Commercial tests are known to have up to 10% Commercial tests are known to have up to 10% false positives false positives (Jalal et al 2007)(Jalal et al 2007)
Screening and confirmatory assay
SLBC 16.10.2008
Sexually transmitted diseasesSexually transmitted diseases Extraction from swab or urineExtraction from swab or urine
125
34
1st step: Extraction from pool of 5 samples (swab or urine).Screening PCR assay.
If pool positive:
2nd step: Rextraction of individual samples and confirmatory PCR assay which uses different targets
1 2 3 4 5
SLBC 16.10.2008
Sexually transmitted diseasesSexually transmitted diseases 460 tested in routine (5,4% positive)460 tested in routine (5,4% positive)
11 Chlamydia11 Chlamydia 10 Mycoplasma10 Mycoplasma 4 Neisseria4 Neisseria
SLBC 16.10.2008
GastroenteritisGastroenteritis Gastroenteritis, also known as stomach flu (although Gastroenteritis, also known as stomach flu (although
unrelated to Influenza)unrelated to Influenza) Caused byCaused by
ParasitesParasites VirusesViruses BacteriaBacteria
Worldwide, gastroenteritis causes the death of 5 to 8 Worldwide, gastroenteritis causes the death of 5 to 8 million people yearlymillion people yearly
Leading cause of death among infants and children Leading cause of death among infants and children under 5under 5
SLBC 16.10.2008
Gastroenteritis: ParasitesGastroenteritis: Parasites Most common:Most common:
Giardia lamblia (often day-care centers, watersupply)Giardia lamblia (often day-care centers, watersupply) Cryptosporidium parvum (Cryptosporidium parvum (diarrhea in children, the diarrhea in children, the
elder and immunocompromised patients) elder and immunocompromised patients) Entamoeba histolyticaEntamoeba histolytica
Advantage of PCR: specificity, no cross-reactions Advantage of PCR: specificity, no cross-reactions
SLBC 16.10.2008
Gastroenteritis: VirusesGastroenteritis: Viruses Caused by:Caused by:
Norovirus (=Caliciviruses) (most common)Norovirus (=Caliciviruses) (most common) Rotavirus (mainly in children)Rotavirus (mainly in children) Adenovirus (mainly in children)Adenovirus (mainly in children) Astrovirus (mainly children)Astrovirus (mainly children)
Viruses are the most common cause of gastroent.Viruses are the most common cause of gastroent.
SLBC 16.10.2008
Parasites/GastrovirusParasites/Gastrovirus Parasitic: one triplex (3 pathogens + IC)Parasitic: one triplex (3 pathogens + IC) Viral: two mulitplexes (5 pathogens + IC)Viral: two mulitplexes (5 pathogens + IC) Important: Internal controlImportant: Internal control 200 mg stool in 1 ml buffer 200 mg stool in 1 ml buffer 10 % inhibition 10 % inhibition 1: 5 dilution 1: 5 dilution less than 1 % inhition, loss of 2 Ct less than 1 % inhition, loss of 2 Ct
1:1 Ct: 33 1:5 Ct: 28
Quantification very difficult
SLBC 16.10.2008
Parasites/GastrovirusParasites/Gastrovirus ParasitesParasites
169 tested in routine (8 % positive)169 tested in routine (8 % positive) 7 Giardia7 Giardia 5 Crypto5 Crypto 1 Entamoeba1 Entamoeba
VirusesViruses 37 tested in routine (19 % positive)37 tested in routine (19 % positive)
2 Adeno2 Adeno 1 Rota1 Rota 4 Noro4 Noro no Astrono Astro
Rest: bacteria or unrelated to pathogensRest: bacteria or unrelated to pathogens
SLBC 16.10.2008
Gastroenteritis: BacteriaGastroenteritis: Bacteria Causes:Causes:
Campylobacter jejuni/coliCampylobacter jejuni/coli Salmonella sp.Salmonella sp. Shigella sp.Shigella sp. Clostridium difficileClostridium difficile Yersinia enterolyticaYersinia enterolytica Escherichia coliEscherichia coli
Enteropathogenic (EPEC)Enteropathogenic (EPEC) eae, bfpeae, bfp genes genes Enteroinvasive (EIEC)Enteroinvasive (EIEC) ipah, invXipah, invX genes genes Entero hemorrhagic (EHEC)Entero hemorrhagic (EHEC) stx1, stx2, hlystx1, stx2, hly genes genes Enterotoxic (ETEC)Enterotoxic (ETEC) STa, STb, LTSTa, STb, LT genes genes Enteroagglomerating (EAEC)Enteroagglomerating (EAEC) aat, eagg, AggRaat, eagg, AggR genes genes
……
SLBC 16.10.2008
Respiratory virusesRespiratory viruses Assay consists of 5 multiplex mixesAssay consists of 5 multiplex mixes Detection of 15 viruses causing respiratory diseasesDetection of 15 viruses causing respiratory diseases
Influenza A and BInfluenza A and B Parainfluenza 1, 2, 3 and 4Parainfluenza 1, 2, 3 and 4 RhinovirusRhinovirus Coronavirus 229, 63 and 43Coronavirus 229, 63 and 43 Respiratory syncitial virus A and BRespiratory syncitial virus A and B Human metapneumovirus A and BHuman metapneumovirus A and B Adenovirus (About 10 % of common cold cases)Adenovirus (About 10 % of common cold cases)
Positive controls as 2 poolsPositive controls as 2 pools Includes Internal ControlIncludes Internal Control Extraction from 1 swab sufficientExtraction from 1 swab sufficient
SLBC 16.10.2008
Additional testsAdditional tests Pathogens are grouped based on clinical Pathogens are grouped based on clinical
symptoms and indicationssymptoms and indications Herpesviruses (HSV1, HSV2, Varizella Zoseter Herpesviruses (HSV1, HSV2, Varizella Zoseter
VirusVirus Fever & Rash (Human Herpesviruses 6 & 7, Fever & Rash (Human Herpesviruses 6 & 7,
Parvovirus B19, Enteroviurs, Measles virusParvovirus B19, Enteroviurs, Measles virus Eye Infections (HSV, VZV, Chlamydia, Eye Infections (HSV, VZV, Chlamydia,
Adenovirus, Enterovirus)Adenovirus, Enterovirus) Meningitis, EncephalitisMeningitis, Encephalitis
SLBC 16.10.2008
Luminex® (up to 100 tests in parallel)Luminex® (up to 100 tests in parallel) More colors, amplification related multiplexing More colors, amplification related multiplexing
problems remainproblems remain Respiratory virusesRespiratory viruses Human Papilloma virus, 15 high risk typesHuman Papilloma virus, 15 high risk types
MicroarraysMicroarrays No amplification necessary?No amplification necessary? Fingerprint of every DNA/RNA moleculae in the Fingerprint of every DNA/RNA moleculae in the
body?body?
Future of multiplexingFuture of multiplexing
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