rare pitfalls associated with the routine use of real...

Rare pitfalls associated with the

routine use of real time PCR

Dr RN Gunson

On Behalf of the West of Scotland Specialist Virology Centre

R+D team

Gartnavel General Hospital

Glasgow

Scotland

Talk outline:

• Glasgow has >10yrs experience of developing

and implementing real time PCR

• Over the years have experienced a number of

rare errors/pitfalls associated with this technique.

• These are VERY VERY RARE

– Some mainly related to use of inhouse protocols

– relevant for users of commercial assays too

Example 1: Strange traces

• You have set up an assay

for the following target:

– RSV (Fam)

• When analysing the post

PCR results the following

results were obtained.

• Any ideas?

Solution

Test with no ROX in pool set up without ROX as passive dye

Example 2: FCV optimisation

• In this experiment you

have carried out an

experiment to optimise a

FCV assay (Cy5)

• When analysing the post

PCR results the following

results were obtained.

Solution

Learning points for commercial kits

• Ensure that the machine is set up correctly

with regards probe and reference dyes.

• The Spectra function on the ABI is useful

for seeing what dyes are present in the

assay.

Example 3: strange traces….

• You have set up an assay

for the following target:

– PF1 (Fam)

– Hmpv (Vic)

– Myco (Cy5)

• Strange flat traces seen

in PF1 assay

These samples are also hMPV

positive!

Multiplexing is limited to a certain number of dyes

Vic being detected in Fam channel

Hints of cross talk

• Flat traces even at strong Ct values

• Positive in another component of the same

test

• Emission dyes with similar spectra:

– Fam/vic

– Vic/NED


• Real time PCR is limited by the number of Dyes available.– 3-4 plex PCR

• Crosstalk is when an increase in flourescence associated with one dye/reporter is also wrongly attributed to another dye/reporter.

• Clues:– Flat traces even at strong Ct values

– Positive in another component of the same test

– Emission dyes with similar spectra:• Fam/vic

• Vic/NED

• Occurs with dyes with similar emission spectra– FAM/Vic

– VIC/Ned

• Problem differs by machine.– ?machines can loose calibration accuracy

IC

Example 4

These are the traces for a dilution series of PF3 tested using a PF3/IC duplex.

All the dilutions also contain an IC at a Ct of 30.

Example 4

This was then repeated from extraction. As you can see the sensitivity of the

reaction seems less than in the previous example. The IC in the second

example had a mean Ct of 26

Test Competition

• Competition for PCR mastermix between two or more different components of an multiplex assay.– the IC and positive target

– Samples containing >1 target.

– Assays that detect and type simultaneously

• The stronger target can be preferentially amplified to the detriment of the weaker target.

• Has numerous effects on test performance

Can reduce sensitivity/flat curves

The presence of the stronger IC in the dilution series on the right

has competed with the PF3 assay resulting in a loss in endpoint

sensitivity and quality of trace.

A B

Can reduce linearity/accuracyThis example is for a BK virus/IC duplex. The example [A] is a dilution series

of BK each containing an IC at a Ct of 25. The example [B] is the same

dilution series this time containing an IC of 30.

Can affect interpretation of the IC

These are the results of the IC component of a dilution series of HCV (ct 16-37).

Each should be positive with a Ct ~27 but in this case less IC has been added

(Ct~30). The stronger the HCV the more affected the IC result

Competition can be overcome.

• Primer limitation experiments– Increases the complexity

– Balance between test sensitivity and competition

– Doesn’t always eliminate the problem entirely.

• Multiplex PCR kits.– Specially designed to amplify >1 target simultaneously.

– RNA and DNA kits available

Single testing

Multiplex assay

(using non multiplex kit)

Multiplex assay

(using mulitplex kit)

Sample Adeno CMV EBV Adeno CMV EBV Adeno CMV EBV

A 31.04 31.34 25.54 31.02 31.19 24.3 32.82 32.65 24.65

B 11.46 28.86 31.30 11 Neg Neg 10.19 29.62 29.62

C 12.12 31.50 30.42 11.02 Neg Neg 10.59 29.28 29.27


• Competition can occur when there is >1 target in the multiplex PCR

– Dual infections

• Can results in loss in sensitivity, trace qualtity and linearity.

• Can complicate IC interpretation.

• In commercial assays (e.g. FTD) the components are carefully balanced and competition should rarely be encounted.

– You may encounter this issue if you add too much IC to the reaction

Example 5:

• We were to implement a

new PCR kit.

– previous work showed it

to be sensitive and

specific

• When the kit was

implemented 6 months

later the following

happened.

PCR kits can lead to false positives


• Carefully assess new kits prior to

implementation

– Endpoint sensitivity

– And specificity.

• Some kits can lead to false positive traces.

• Some can be related to a particular batch

Example 6: HCV quantification

• You are setting up a HCV quantification PCR.– 76 samples

– 10 standards (plasmid)

– 1 positive control (plasmid)

– 300 IU/ml extraction control (sample)

• Post PCR results:– Samples, IC and 300 IU

control are negative.

– Standards/positive control work well


• Controls based on plasmids can work

despite failed extraction or RT.

– Be aware of what your controls are

– Always interpret along side IC

Example 7: H1N1 panic

• On Saturday April

25th, a couple

returned from Mexico

with “flu-like illness”

– 2 samples

– Flu A positive

– H1, H3 and H5

negative

Swine/avian H1N1

Seasonal H1N1

H1N1 sw

H1N1 reference strain

1st 2 Scottish patients

Example 7: H1N1 panic

Following this:

• 45 respiratory samples taken from– Contacts

– others travelling from US/Mexico.

• Tested using the following triplex:– Flu A (Fam)

– Flu B (Vic)

– RSV (Cy5)

• Large number of positive traces.– Wrong controls were positive

– ?controls in wrong order

• Repeat testing failed to confirm these findings.

– All were negative

Spectra of the 1st test

Spectra of the second test

Solution

• The wrong test was used on the first occasion:

– Clue: the controls did not work

– Spectra was wrong.

• Further investigation revealed that the test

incorporating Rhino, RSV and adenovirus was

used by accident.

– The positive traces were all rhinovirus positive and

not flu A.

– Panic over……..


• Be careful when setting up PCR to use the

correct p/p tubes.

• It can be easy to use the wrong tube

(unless they are clearly differentiated).

• Controls and examination of the spectra

will aid you in your troubleshoot.

Example 8: Mutant H1N1

Flu A season 2010

• Number of samples that were (n=16) Flu A negative but swine flu positive.

• ?virus changed– Implications for our test

• All positive controls had worked as expected– Except positive swine flu result

in the rhinovirus control.

• Repeating the test failed to confirm the extra swine flu positive samples

Our Flu A/rhinovirus test has this spectra

Solution

• The flu A, swine flu, H275Y primer probe had been mixed with the Flu A, rhinovirus primer probe.– Strangely both tests worked.

– Proven by examination of the spectra

• All the swine flu positive/flu A negative samples were actually rhinovirus positives.– Swine flu and rhinovirus are both labelled with VIC dye.

– Explains why the Flu A test failed to detect these as positive.

• The detection of swine flu in the rhinovirus control was the clue as to the error…– Showed that the PCR assay contained rhinovirus primers/probe.


• Be careful when setting up PCR to use the

correct p/p tubes.

• It can be easy to use the wrong tube

(unless they are clearly differentiated).

• Controls and spectra examination will aid

you in your troubleshoot.

Example 9: mutant h1n1

Learning points for commecial kits

• Rarely mutations in the test target can

lead to test failures

• But this is very rare…….

Example 10: HCV

• A new lot of HCV primer/probe pool was manufactured and checked in Dec 2010.

• It was introduced in February 2011.– False positive in all tests

– Including NTC

• New HCV reagents were negative


• PCR reagents can degrade despite being

properly stored.

Example 11: HBV issue

• Set up a qPCR for HBV

• Positive controls/standards worked

• 7 out of 8 negative controls were positive

• Large number of weak positive samples

• NTC was negative

hbv negatives

Solution

• NTC was negative

– Suggests that the reagents are not degrading

or contaminated

• Only change to protocol was recent

introduction of new lot of mCMV internal

control

– grown in BHK cell

Solution

• The most recent stock of mCMV (undiluted) was extracted and tested for HBV– Positive (ct ~20)

• Further examination revealed that it had been grown in a different cell line (PLC/PRF/5).

Rare triple infections

• During 2010-11 influenza season we detected a number of samples (n=23) that contained – Influenza A (H3)

– Influenza B

– Influenza A (H1N1)

• All three viruses were circulating.

• ?contamination.

What did we do?

• Clean up of laboratory surfaces

• Checked reagents were “clean”

• All results repeatable from extraction.

Learning points

• Important to rule out laboratory contamination

– Negative controls

– Check reagents with “water” runs

– Check all set up areas

– Check extraction machines with water runs

• Contamination can occur before samples even reach the laboratory.

Example 12: B19

• We were using a PCR for B19

• In order to quantitate we ordered a full length DNA oliognucleotide representing the amplicon of a B19 real time PCR assay

• The test worked well for a number of months.

• ~6 months later a new lot of primer/probe pool was introduced and found to be contaminated with B19.

Solution

• The new primers and probe were ordered from the same manufacturer as who made the oligo.

• ?contaminated at manufacture ? In laboratory

• An alternative sythesis of primers/probe were obtained from another supplier and were negative for B19

• Also seen for other tests (e.g. norovirus, HIV)– Sometimes related to reagent manufacture

– Sometime related to mastermix

– Sometime seen in EQA panels.

• Follow up: we reorder primers and probe from the original manufacturer 2 years later and B19 contamination could still be detected.

Learning points

• Important to rule out laboratory contamination

– Negative controls

– Check reagents with “water” runs

– Check all set up areas

– Check extraction machines with water runs

• Contamination can occur before samples even reach the laboratory.

Summary

• Multiplex real time PCR has many

advantages.

• These VERY rare issues are useful to

know about

– Hopefully you wont encounter them

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