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