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

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The basics of PCR: Common groundThe basics of PCR: Common groundBacteria

Parasites

VirusesHuman

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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

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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

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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

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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)

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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

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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

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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

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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

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Real-time PCR: the TaqMan® assayReal-time PCR: the TaqMan® assay

Q

R

Quencher

Reporter dye (fluorophore)

2

3

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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

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Multiplex PCR: Limitation, spectral Multiplex PCR: Limitation, spectral overlappingoverlapping

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Real-time PCR: equipmentReal-time PCR: equipment

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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

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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

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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

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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

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HCV duplex assay: ExampleHCV duplex assay: Example

HCV (FAM)BMV (YAK)

4 HCV positive patients

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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

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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

……

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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

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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

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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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AcknowledgementsAcknowledgementsBen WeberUdo MargraffVera HungerNatascha van der TaelemAlain MenzelFréderique ParisotSandra MotschKerstin Unger

Miriam SteimerPriscilla PetitFlorence Caillol