overview multi pcr

Theo Sloots

Queensland Paediatric Infectious Diseases Laboratory

SASVRC, Royal Children’s Hospital

Multiplex PCR – overview and limitations

Conventional PCR-ELISA

• Influenza A & B; Para 1,2 & 3; RSV & Adenovirus• Plasmodium falciparum,P. Malariae, P. Ovale & P. Vivax• Polyomaviruses JCV & BKV• HSV types 1 & 2; VZV

Real-time PCR

• Bartonella species & B. hensalae• Bordetella pertussis & Bordetella parapertussis,• Legionella species & L. pneumophila • Neisseria gonorrhoeae, Chlamydia trachomatis & internal control• Neisseria meningitidis (por A; ctrA)• HSV types 1 & 2• Influenza A & B• Para 1,2 & 3 • RSV & Adenovirus• Plasmodium falciparum & P. Vivax • P. Malariae & P. Ovale • Polyomaviruses JCV & BKV

QPID lab’s multiplex assays include:

Multiplex assays – the basics:

• Multiplex PCR is a variant of PCR in which two or more targets are simultaneously amplified in the same reaction.

• Has been successfully applied in many areas of DNA testing including molecular diagnosis of infectious disease

• Many factors can influence the results of multiplex analysis.

Staphylococcus aureus GenomeStaphylococcus aureus Genome

nuc

• Detection of genus-specific gene sequences

- nuc gene is specific for ALL Staph aureus

16s rRNA

• Detection of 16s rRNA gene - common to ALL bacteria• Detection of 16s rRNA gene - common to ALL bacteria

APPLICATION OF MxPCR TO ANTIBIOTIC RESISTANCEAPPLICATION OF MxPCR TO ANTIBIOTIC RESISTANCE

• Detection of drug resistance

- mec A gene confers methicillin resistance in MRSA

• Detection of drug resistance

- mec A gene confers methicillin resistance in MRSA

mec A

Conventional Multiplex assays – the basics:

• Agarose gel electrophoresis: PCR product size

100bp

200bp

300bp

(1) (2) (3)

400bp

(3) Multiplex

(1)

(2)

(3)


• Multiplex PCR is a variant of PCR in which two or more targets are simultaneously amplified in the same reaction.

• Has been successfully applied in many areas of DNA testing including molecular diagnosis of infectious disease using conventional and real-time PCR

• Many factors can influence the results of multiplex analysis.

• Amplification of the different templates can be detected in a number of ways:

• Conventional PCR - agarose gel detection- solid phase hybridisation with colour detection

• Real-time PCR - fluorescent probes

Application: PCR-ELAHA: Respiratory virus multiplex

Reaction Mix:• 7 primer pairs - Influenza A

- Influenza B- Parainfluenza 1- Parainfluenza 2- Parainfluenza 3- RSV- Adenovirus

• One Step RT-PCR kit (Invitrogen)

• DIG-11-dUTP

ELAHA Detection (Enzyme Linked Amplicon Hybridisation Assay):

•• 7 biotinylated probes (one specific for each virus)• streptavidin coated microtitre plate• anti-DIG antibody• TMB substrate

(Syrmis et al. J Mol Diagn. 2004 May;6(2):125-31.)


solid phase hybridisation with colour detection

• PCR-ELISA: biotinylated specific probes

3.46

0.03

0.03

0.04

0.03

2.81

2.97

3.12

(1) (2) (3)

Template 1

Template 2

Target is amplifiedDIG is incorporated

Microtitre plateis coated with specific probe

Amplicon is captured

(hybridisation)

Hybrid is detected(anti-DIG)

11 22 33 44

(DIG = digoxigenin)

Specific probe & well for each virus:

InfA InfB AdenoRSVpara3para2para1

(Syrmis et al. J Mol Diagn. 2004 May;6(2):125-31.)

InfA

InfB

Adeno

RSV

para3

para2

para1

InfA

InfB

Adeno

RSV

para3

para2

para1

Positive controls:

Probes:

Real-time PCR platforms:

Real-time multiplex PCR

Limited to 4 (or 6) detection channels


• Two different methods of detecting multiple targets using real-time PCR are commonly used:

Eg.� Agarose gel electrophoresis: PCR product size� PCR-ELISA: biotinylated specific probes� Real-time PCR: fluorophore labeled specific probes� Real-time PCR: melting curve analysis

Common Real-time PCR detection chemistries:

Adjacent Hybridisation Probes (HybProbes)

TaqMan (Nuclease) probes3’5’

3’5’ 5’ 3’

Adjacent Hybridisation Probes (HybProbes):

Light source

480nm

F1510-550nm

F2615-675nm

F3670-750nm

Detection channels (Roche LightCycler)

3’

5’

5’

3’

Probe 2

Probe 1

LCRed-640 (acceptor)fluorescein (donor)


Light source

480nm

F1510-550nm

F2615-675nm

F3670-750nm


PCR product

3’5’ 5’ 3’

Probe 2Probe 1

fluorescein (donor) LCRed-640 (acceptor)


Light source

480nm

F1510-550nm

F2615-675nm

F3670-750nm


FRETFRET

PCR product

3’5’ 5’ 3’

Probe 2Probe 1



APPLICATION: N.gonorrhoeae / C.trachomatis (duplex; LightCycler)

Primers:gono-F CGGTTTCCGTGCGTTACGAgono-R CTGGTTTCATCTGATTACTTTCCAChlam-F CTGCTTCCTCCTTGCAAGCT Chlam-R ACGCATGCTGATAGCGTCA

Hybridisation probes:gono-P1 CATTCAATTTGTTCCGAGTCAAAACAGC–fl. gono-P2 LCred640–AGTCCGCCTATACGCCTGCTACTTTCAC–Ph.Chlam-P1 TTCCCACAGAATTCCGTCGATCATAA–fl. Chlam-P2 LCred705–CTTGGTTCAGCAGGATTCCCCAC–Ph.

(Whiley DM, Sloots TP. Pathology. 2005 Oct;37(5):364-70.)

NG (F2) CT (F3)

Melting Curve Analysis:

• Primers homologous to HSV-1 and HSV-2 DNA• Probe 2 has sequence mismatches with HSV-2 genome• This enables HSV-1 and HSV-2 to be distinguished by

Melting Curve analysis

Whiley et al. J Clin Micro. 2001;39:4357-4361.

herpes simplex virus DNA – DNA pol

PROBE 1PROBE 1 PROBE 2PROBE 2

CTGAGGCTGCTGCTGGCCACAGGATTTTCTGAGGCTGCTGCTGGCCACAGGATTTT AAGTAGCTGAAATTGCTGCTGGAGAGGCTGCTGTAGCTGAAATTGCTGCTGGAGAGGCTGCT

HSVHSV--1 GACTCCGACGACGACCGGTGTCCTAAAA 1 GACTCCGACGACGACCGGTGTCCTAAAA TCATCGACTTTAACGACGACCTCTCCGACGA

PROBE 1PROBE 1 PROBE 2PROBE 2

CTGAGGCTGCTGCTGGCCACAGGATTTTCTGAGGCTGCTGCTGGCCACAGGATTTT AAGTGTAAGCTGAAATTGCTGCTGGGCTGAAATTGCTGCTGGAAGAGGCTGCTGAGGCTGCT

HSVHSV--2 GACTCCGACGACGACCGGTGTCCTAAAA 2 GACTCCGACGACGACCGGTGTCCTAAAA TCAGCGACTTTAACGACGACCCCTCCGACGA

Primer Binding Site (s)

Primer Binding Site (as)

Probe Binding Sites

TaqMan (Nuclease) probes

Light source FAM CY5

Detection channels (ABI7500)

3’5’

BHQ1 (quencher)FAM (reporter)

JOE

3’5’


FRETFRET

Light source FAM JOE CY5


TaqMan probes :

3’5’


PCR product



JOE

TaqMan probes :

3’5’


PCR product

Primer



JOE

TaqMan probes :

3’5’


PCR product

DNA polymerase

Primer extension



JOE

TaqMan probes :

3’

BHQ1 (quencher)

FAM (reporter)

PCR product

DNA polymerase

Primer extension



JOE

TaqMan probes :

3’

BHQ1 (quencher)

JOE (reporter)

PCR product

DNA polymerase

Primer extension



JOE

TaqMan probes :

3’

BHQ3 (quencher)

CY5 (reporter)

PCR product

DNA polymerase

Primer extension



JOE

TaqMan probes :

TaqMan probes :

Eg. N.gonorrhoeae, C.trachomatis, internal control (triplex; ABI)

Primers:gono-F CAGCATTCAATTTGTTCCGAGTC gono-R GAACTGGTTTCATCTGATTACTTTCCAChlam-F CCACAGAATTCCGTCGATCA Chlam-R TGCCGCTTTGAGTTCTGCTTIC-F CATGGGAAGCAAGGGAACTAATGIC-R CCCAGCGAGCAATACAGAATTT

TaqMan probes:

gono-TM FAM–CGCCTATACGCCTGCTACTTTCACGC–BHQ1

Chlam-TM JOE–ATTCCCCACAGGCAGAGCTTGCAA–BHQ1

IC-TM CY5–TCTTCCCTCGAACCTGCACCATCAAGTCA–BHQ3

(Whiley DM, Sloots TP. Pathology. 2005 Oct;37(5):364-70.)

NG (FAM) CT (JOE) IC (CY5)

Benefits & Limitationsof multiplex PCR

Multiplex PCR assays:

BENEFITS –

• Reduced cost

• Reduced hands-on-time

� fewer reaction mixes to make, store, QC etc.� Fewer reactions per sample to prepare

• Higher throughput

� saves valuable space on real-time PCR instrumentation

PlatformPlatform 3 x monoplex3 x monoplex Multiplex (x3)Multiplex (x3)

LightCyclerLightCycler $ 9.75$ 9.75 $ 4.00$ 4.00

ABI/RotorGeneABI/RotorGene $ 6.90$ 6.90 $ 3.05$ 3.05

Multiplex PCR assays:

LIMITATIONS –

• Preferential amplification of one target sequence over another is a known phenomenon in multiplex PCR (ie bias in template-to-product ratio)

• Two processes that induce this bias have been identified,

• PCR drift and PCR selection (competitive inhibition)

PCR drift

PCR drift is due to stochastic fluctuation in the interactions of PCR reagents particularly in the early cycles, which could arise in the presence of very low template concentrations, or through assay design.

Eg. Primer / probe interactions:

EXAMPLE:

Duplex LC assay for Bordetella pertussis & B. parapertussis: � sensitivity was reduced when assays were duplexed.

Detection limit (copies/reaction)B. pertussis B. parapertussis

------------------------------------------------------------------------Individual assays 10 10Duplex 100 1000------------------------------------------------------------------------

(Kosters et al. J Clin Microbiol. 2002 May;40(5):1719-22.)

B.pertussis probe2

B.parapertussis reverse primer

Primer Premier Software:

� The loss of sensitivity for the B.parapertussis assay was caused by interaction between the reverse primer and the 2nd

hydrolysis probe of B.pertussis.

The 3’ end of the B.parapertussis primer was complementary to the B.pertussis probe.

1.Primer/probe binding limits their availability during the reaction2.The primer can bind to the probe and extend, reducing the available primer for amplification of B. parapertussis DNA.

It is important to carefully select primers and probes.

PCR selection (competitive inhibition)

PCR selection is defined as a mechanism which inherently favours the amplification of certain templates due to the relative target concentrations or properties of the target.

• Amplification bias may also be due to the choice of primers used in the multiplex PCR.

• Primer pairs with high amplification efficiency will produce amplification product independent of starting template concentrations.

• Primers with lower amplification efficiency can result in amplification bias depending on the template (concentration)

PCR selection (competitive inhibition)

• In cases of mixtures of primer pairs of high and low efficiency, the earlier amplification of one target may inhibit the amplification of a second target

• Can be an issue for inclusion of internal controls:

� internal control DNA added to a reaction mix should be less than the expected Target DNA

This Means That:

QUESTION:

Is competitive inhibition influenced by assay design ?

In particular:

Examine consensus (LightCycler) PCR versus type-specific (TaqMan) PCR

ASSAY: Detection and differentiation of HSV-1 and HSV-2

•Compared consensus primers and hybridisation probes followed by melting curve analysis

to

•Type-specific primers and TaqMan probes in a duplex assay

HSV-1 DNA – DNA pol

DUPLEX: HSV-1 and HSV-2 -specific primers and (TaqMan) probes:

Primers and probes are specific for HSV-1Primers and probes are specific for HSV-2

Consensus primers and LC probes:

HSV-1 and/or HSV-2 DNA (DNA pol)

Primers are common (consensus) for HSV-1 and

HSV-2 sequences

LC probes have mismatches for HSV-2

Primer 1

Primer 2

LC Probes

HSV-2 ProbeHSV1 Primer 1

HSV1 Primer 2

HSV-1 Probe HSV2 Primer 1

HSV2 Primer 2

HSV-2 DNA – DNA pol

Detection of HSV types 1 and 2

Dilutions: 1 2 3 4 5 6

Copies of HSV type 1 107 106 105 104 103 102

Copies of HSV type 2 104 104 104 104 104 104

Results:

Hybridisation probe assayHSV type 1 POS POS POS POS POS negHSV type 2 neg neg POS POS POS POS

Duplex TaqMan probe assayHSV type 1 POS POS POS POS POS POSHSV type 2 POS POS POS POS POS POS

Consensus primer sequences:

Note:

• Where a consensus primer pair can amplify 2 different targets, and both targets are present in a specimen, the PCR will favour the target at greatest concentration.

• Generally will only detect both targets when their relative difference in concentration does not exceed one log of concentration.

• If the detection of a particular viral type carries special clinical importance then type-specific primers and probes should be used in preference to consensus oligonucleotide sequences.

Whiley DM, Sloots TP. Pathology. 2005 Jun;37(3):254-6

• OR - is it best to use individual assays for some targets: ie. not multiplex.

QUESTION:

Is the duplex assay using target-specific primers and probes influenced by differences in target copy number ?

Monospecific versus DUPLEX PCR: Competitive Inhibition

Four dilutions of N.gono were prepared:

1x105 copies1x104

1x103

1x102

Each NG dilution spiked with 2x103 CT DNA

(Ct=29)

All samples were then tested in a monospecific assay for NG and a duplex

assay for NG and CT

15

20

25

30

35

40

10E5 10E4 10E3 10E2

Dilution

Ct

Valu

e

N.gono only PCRN.gono only PCR105

104

103

102

Monospecific primers and TaqMan

probe for NG

JC/BK duplexJC/BK duplex

Presence of both NG and CT in the test sample

affects the amplification efficiency of both assays

Monospecific primers and

TaqMan probes for NG & CT

NG:CT DuplexNG:CT Duplex

20

25

30

35

40

45

10E5 10E4 10E3 10E2

Copies

Ct

Va

lue


15

20

25

30

35

40

10E5 10E4 10E3 10E2

Dilution

Ct

Valu

e

N.gono only PCRN.gono only PCR105

104

103

102

Monospecific primers and TaqMan

probe for NG

JC/BK duplexJC/BK duplex

Presence of both NG and CT in the test sample

affects the amplification efficiency of both assays

Monospecific primers and

TaqMan probes for NG & CT


NG =105

CT=2x103Ct=+0.5

• In the Duplex assay, the amplification efficiency of NG PCR decreased as the ratio of NG:CT decreased

• The target in excess was amplified with greater efficiency.

• The Ct value (threshold of amplification) increased with diminishing ratio of NG:CT


CT =2x103

NG=102

CT =2x103

NG=103


The relative concentration of two The relative concentration of two

(or more) targets is important for (or more) targets is important for

amplification efficiencyamplification efficiency

In extreme cases the target in low In extreme cases the target in low

concentration may be missedconcentration may be missed

CASE STUDY: C. trachomatis false-negative result

• Urine specimen: (Commercial NG/CT/IC multiplex NAAT)� Result: NG positive; CT negative

• Patient treated for NG only

• Patient remained symptomatic

• Urine retested using individual NG & CT real-time PCRs:

� NG positive; CT positive� [NG DNA] approx. 10,000 times greater than [CT DNA]

CONCLUSION:

Competitive inhibition of CT reaction caused false negative result in commercial assay.

Multiplex PCR Assay Design(in brief)

•The same design principles apply to Multiplex PCR as for PCR of a single target:

•The first few rounds of thermal cycling have a substantial effect on the overall sensitivity and specificity of PCR

•Assuming efficient denaturation of the target, overall success of specific amplification depends on

• the rate at which primers anneal to their target • the rate at which annealed primers are extended along

the target sequence.

Multiplex PCR Assay DesignMultiplex PCR Assay Design

� Thus, the majority of modifications to improve multiplex PCR performance have been directed towards the factors affecting annealing and/or extension rates.

oThe presence of more than one primer/probe pair in the multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of dimers

oThese nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension.

oThus, the optimization of multiplex PCR should aim to minimize or reduce such nonspecific interactions.


• Ideally, all the primer pairs in a multiplex PCR should enable similar amplification efficiencies for their respective target.

• Pay special attention to primer design parameters such as

• homology of primers with their target nucleic acid sequences, • their length, • the GC content, • and their concentration

• This may be achieved through the utilization of primers with nearly identical optimum annealing temperatures. (primer length of 18 to 30 bp; a GC content of 35 to 60%)

• Primers should not display significant homology either internally or to one another


This is often a process of trial and error

– going back and forth from sequence data to design software.- testing of primers to redesign

Use Design software:

� Primer Express (initial primer/probe design)

� Primer Premier (multiplex assays)


Select primer sequences

Test/align primer sequences

Optimise by

conventional monoplex PCR

Multiplex PCR

Equimolar primer mix

(A) All Products are Weaka.use longer extension

b.decrease extension tempc.decrease annealing temp

d.adjust Taq concentratione.combine Aa, Ab, Ac, Ad

(C) Short Products are Weaka.increase buffer concentration

b.decrease anneal and/or exten tempc.increase amt primer for weak loci

d.Combine Ca, Cb, Cc

(B) Long Products are Weaka.use longer extension

b.increase anneal and/or exten tempc.increase amt primer for weak loci

d.decrease buffer concentration(keep Mg conc constant)

e.Combine Ba, Bb, Bc, Bd

(D) Non-specific Products Appeara.Long-increase buffer concentration

b.Short-decrease buffer concentrationc.increase annealing temperature

d.decrease template and Taqe.Increase Mg concentration

f.Combine Da, Db, Dc, Dd, De

(E) None of the Above Worksa.Use adjuvant - BSA

b.Use adjuvant – DMSO or glycerolc.Check primers for interactions

d.Prepare new reagentse.Use fresh dNTPs

Final Considerations

• Other issues:

� Involves an extensive optimisation process eg. must balance reaction parameters to enable all targets to

amplify while not affecting the sensitivity of each test.

� Requires special consideration for Quality control and Quality Assurance (multiple target controls)

• NPAAC Guidelines:

� The validation, QC etc. of multiplex PCR assays is not specifically covered in the current NPAAC Guidelines.

� Given the special issues surrounding multiplex PCR, guidelines for these may need to be developed.


overview multi pcr

Documents

agarose gel

solid phase

melting curve

increase amt

increase buffer

decrease buffer

adjacent hybridisation

test sample