Rare pitfalls associated with the routine use of real time PCR

Transcription

1 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

2 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

3 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?

4 Solution

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

6 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.

7 Solution

8 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.

9 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

10 These samples are also hmpv positive!

11 Multiplexing is limited to a certain number of dyes

12 Vic being detected in Fam channel

13 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

14 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

15 Example 4 These are the traces for a dilution series of PF3 tested using a PF3/IC duplex. IC All the dilutions also contain an IC at a Ct of 30.

16 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

17 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

18 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.

19 Can reduce linearity/accuracy This 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. A B

20 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

21 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 B Neg Neg C Neg Neg

22 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

23 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.

24 PCR kits can lead to false positives

25 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

26 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

27 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

28 Example 7: H1N1 panic On Saturday April 25 th, a couple returned from Mexico with flu-like illness 2 samples Flu A positive H1, H3 and H5 negative

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30 Swine/avian H1N1 1st 2 Scottish patients H1N1 sw H1N1 reference strain Seasonal H1N1

31 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

32 Spectra of the 1 st test

33 Spectra of the second test

34 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..

35 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.

36 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

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39 Our Flu A/rhinovirus test has this spectra

40 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.

41 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.

42 Example 9: mutant h1n1

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44 Learning points for commecial kits Rarely mutations in the test target can lead to test failures But this is very rare.

45 Example 10: HCV A new lot of HCV primer/probe pool was manufactured and checked in Dec It was introduced in February False positive in all tests Including NTC New HCV reagents were negative

46 Learning points for commercial kits PCR reagents can degrade despite being properly stored.

47 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

48 hbv negatives

49 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

50 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).

51 Rare triple infections During 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.

52 What did we do? Clean up of laboratory surfaces Checked reagents were clean All results repeatable from extraction.

53 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.

54 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.

55 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.

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58 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.

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60 Summary Multiplex real time PCR has many advantages. These VERY rare issues are useful to know about Hopefully you wont encounter them