Multiplex PCR overview and limitations

Theo Sloots
Queensland Paediatric Infectious Diseases Laboratory SASVRC, Royal Childrens Hospital

QPID labs multiplex assays include:

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

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.

APPLICATION OF MxPCR TO ANTIBIOTIC RESISTANCE Staphylococcus aureus Genome

16s rRNA nuc mec A

Detection of 16s rRNA gene - common to ALL bacteria

Detection of genus-specific gene sequences - nuc gene is specific for ALL Staph aureus Detection of drug resistance - mec A gene confers methicillin resistance in MRSA

Conventional Multiplex assays the basics: Agarose gel electrophoresis: PCR product size

(1)

(2)

(3) (3)

Multiplex

400bp 300bp 200bp 100bp

(1) (2) (3)

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 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: Reaction Mix:

PCR-ELAHA: Respiratory virus multiplex

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.)

Multiplex assays the basics:

solid phase hybridisation with colour detection

PCR-ELISA: biotinylated specific probes

1
Target is amplified DIG is incorporated
(DIG = digoxigenin)

2
Microtitre plate is coated with specific probe

3
Amplicon is captured (hybridisation)

4
Hybrid is detected (anti-DIG)

(1)
3.46

(2)
0.03

(3)
2.97 0.03

Template 1 Template 2

0.03

2.81

3.12

0.04

Specific probe & well for each virus:

InfA

InfB

para1

para2

para3

RSV

Adeno

para1

para2

para3

Adeno

RSV

InfA

InfA InfB para1 para2 para3 RSV Adeno

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

InfB

Positive controls:

Real-time multiplex PCR

Real-time PCR platforms:

Limited to 4 (or 6) detection channels

Multiplex assays the basics:

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)

5 3 5 3

TaqMan (Nuclease) probes

Adjacent Hybridisation Probes (HybProbes):

fluorescein (donor)
5 Probe 1

LCRed-640 (acceptor)
3 Probe 2

Light source
480nm

F1

F2

F3

510-550nm 615-675nm 670-750nm Detection channels (Roche LightCycler)

Adjacent Hybridisation Probes (HybProbes): fluorescein (donor)

5 Probe 1 Probe 2 3 5

LCRed-640 (acceptor)
3

Light source
480nm

F1

F2

F3

510-550nm 615-675nm 670-750nm Detection channels (Roche LightCycler)

Adjacent Hybridisation Probes (HybProbes):

fluorescein (donor)

LCRed-640 (acceptor)

Probe 1 5 3 5

Probe 2 3

PCR product

Light source
480nm

F1

F2

F3

510-550nm 615-675nm 670-750nm Detection channels (Roche LightCycler)

Adjacent Hybridisation Probes (HybProbes):

fluorescein (donor)

FRET

LCRed-640 (acceptor)

Probe 1 5 3 5

Probe 2 3

PCR product

Light source
480nm

F1

F2

F3

510-550nm 615-675nm 670-750nm Detection channels (Roche LightCycler)

Adjacent Hybridisation Probes (HybProbes):

fluorescein (donor)

FRET

LCRed-705 (acceptor)

Probe 1 5 3 5

Probe 2 3

PCR product

Light source
480nm

F1

F2

F3

510-550nm 615-675nm 670-750nm Detection channels (Roche LightCycler)

Adjacent Hybridisation Probes (HybProbes):

APPLICATION: N.gonorrhoeae / C.trachomatis (duplex; LightCycler)
Primers: gono-F gono-R Chlam-F Chlam-R CGGTTTCCGTGCGTTACGA CTGGTTTCATCTGATTACTTTCCA CTGCTTCCTCCTTGCAAGCT ACGCATGCTGATAGCGTCA

Hybridisation probes: gono-P1 CATTCAATTTGTTCCGAGTCAAAACAGCfl. gono-P2 LCred640AGTCCGCCTATACGCCTGCTACTTTCACPh. Chlam-P1 TTCCCACAGAATTCCGTCGATCATAAfl. LCred705CTTGGTTCAGCAGGATTCCCCACPh. Chlam-P2

NG (F2)

CT (F3)

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

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

Probe Binding Sites Primer Binding Site (s)

Primer Binding Site (as)

herpes simplex virus DNA DNA pol

PROBE 1 PROBE 2 CTGAGGCTGCTGCTGGCCACAGGATTTT AGTAGCTGAAATTGCTGCTGGAGAGGCTGCT HSV-1 GACTCCGACGACGACCGGTGTCCTAAAA TCATCGACTTTAACGACGACCTCTCCGACGA PROBE 1 PROBE 2 CTGAGGCTGCTGCTGGCCACAGGATTTT AGTAGCTGAAATTGCTGCTGGAGAGGCTGCT HSV-2 GACTCCGACGACGACCGGTGTCCTAAAA TCAGCGACTTTAACGACGACCCCTCCGACGA

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

TaqMan (Nuclease) probes

FAM (reporter)

BHQ1 (quencher)

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) BHQ1 (quencher)

FRET

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) BHQ1 (quencher)

5
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) BHQ1 (quencher)

Primer
5
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) DNA polymerase Primer extension

5
PCR product

BHQ1 (quencher)

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) BHQ1 (quencher) DNA polymerase Primer extension
3
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : FAM (reporter) BHQ1 (quencher) DNA polymerase Primer extension
3
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : JOE (reporter) BHQ1 (quencher) DNA polymerase Primer extension
3
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

TaqMan probes : CY5 (reporter) BHQ3 (quencher) DNA polymerase Primer extension
3
PCR product

Light source

FAM

JOE

CY5

Detection channels (ABI7500)

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

Primers: gono-F CAGCATTCAATTTGTTCCGAGTC gono-R GAACTGGTTTCATCTGATTACTTTCCA Chlam-F CCACAGAATTCCGTCGATCA Chlam-R TGCCGCTTTGAGTTCTGCTT IC-F CATGGGAAGCAAGGGAACTAATG IC-R CCCAGCGAGCAATACAGAATTT TaqMan probes: FAMCGCCTATACGCCTGCTACTTTCACGCBHQ1 gono-TM Chlam-TM JOEATTCCCCACAGGCAGAGCTTGCAABHQ1 IC-TM CY5TCTTCCCTCGAACCTGCACCATCAAGTCABHQ3

NG (FAM)

CT (JOE)

IC (CY5)

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

Benefits & Limitations of multiplex PCR

Multiplex PCR assays:

BENEFITS Reduced cost
Platform LightCycler ABI/RotorGene 3 x monoplex $ 9.75 $ 6.90 Multiplex (x3) $ 4.00 $ 3.05

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

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 10 Duplex 100 1000 -----------------------------------------------------------------------(Kosters et al. J Clin Microbiol. 2002 May;40(5):1719-22.)

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.

Primer Premier Software:

B.parapertussis reverse primer B.pertussis probe2

The 3 end of the B.parapertussis primer was complementary to the B.pertussis probe.
1.Primer/probe binding limits their availability during the reaction 2.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) This Means That:

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

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

Consensus primers and LC probes:

LC Probes
Primer 1

Primers are common (consensus) for HSV-1 and HSV-2 sequences LC probes have mismatches for HSV-2

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

Primer 2

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

HSV1 Primer 1

HSV-1 Probe

HSV2 Primer 1

HSV-2 Probe

HSV-1 DNA DNA pol

HSV1 Primer 2

HSV-2 DNA DNA pol

HSV2 Primer 2

Primers and probes are specific for HSV-1

Primers and probes are specific for HSV-2

Detection of HSV types 1 and 2

Dilutions: Copies of HSV type 1 Copies of HSV type 2 Results: Hybridisation probe assay HSV type 1 HSV type 2 Duplex TaqMan probe assay HSV type 1 HSV type 2

1 107 104

2 106 104

3 105 104

4 104 104

5 103 104

6 102 104

POS neg

POS neg

POS POS

POS POS

POS POS

neg POS

POS POS

POS POS

POS POS

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

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

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

Monospecific versus DUPLEX PCR: Competitive Inhibition

Four dilutions of N.gono were prepared: 1x105 copies 1x104 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

N.gono only PCR

40 35 Ct Value 30 25

105 104 103 102

20

Monospecific primers and TaqMan probe for NG

10E5 10E4 Dilution 10E3 10E2

15

NG:CT Duplex JC/BK duplex

Monospecific primers and TaqMan probes for NG & CT Presence of both NG and CT in the test sample affects the amplification efficiency of both assays

Monospecific versus DUPLEX PCR: Competitive Inhibition

N.gono only PCR

40 35 Ct Value 30 25

105 104 103 102

45

20

Monospecific primers and TaqMan probe for NG

10E5 10E4 Dilution 10E3 10E2

15

40

Ct Value

35

30

25

NG:CT Duplex JC/BK duplex

Monospecific primers and TaqMan probes for NG & CT

20 10E5 10E4 Copies 10E3 10E2

Presence of both NG and CT in the test sample affects the amplification efficiency of both assays

NG:CT Duplex
NG =105

Monospecific versus DUPLEX PCR: Competitive Inhibition

Ct=+0.5

CT=2x103

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

NG=103

CT =2x103

CT =2x103

NG=102

The relative concentration of two (or more) targets is important for amplification efficiency In extreme cases the target in low concentration 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)

Multiplex PCR Assay Design

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.

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

Multiplex PCR Assay Design

o The 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 o These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. o Thus, the optimization of multiplex PCR should aim to minimize or reduce such nonspecific interactions.

Multiplex PCR Assay Design

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

Multiplex PCR Assay Design

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 Weak

a.use longer extension b.decrease extension temp c.decrease annealing temp d.adjust Taq concentration e.combine Aa, Ab, Ac, Ad

(B) Long Products are Weak

a.use longer extension b.increase anneal and/or exten temp c.increase amt primer for weak loci d.decrease buffer concentration (keep Mg conc constant) e.Combine Ba, Bb, Bc, Bd

(C) Short Products are Weak

a.increase buffer concentration b.decrease anneal and/or exten temp c.increase amt primer for weak loci d.Combine Ca, Cb, Cc

(D) Non-specific Products Appear

a.Long-increase buffer concentration b.Short-decrease buffer concentration c.increase annealing temperature d.decrease template and Taq e.Increase Mg concentration f.Combine Da, Db, Dc, Dd, De

(E) None of the Above Works

a.Use adjuvant - BSA b.Use adjuvant DMSO or glycerol c.Check primers for interactions d.Prepare new reagents e.Use fresh dNTPs

Multiplex PCR Assay Design

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.