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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
Learning points for commercial kits
• 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
Learning points for commercial kits
• 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
Learning points for commercial kits
• 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
Learning points for commercial kits
• 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……..
Learning points for commercial kits
• 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.
Learning points for commercial kits
• 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
Learning points for commercial kits
• 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