Quantitative PCR (real time PCR) Kristin Elwin Cryptosporidium Reference Unit, Wales, UK

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1 Quantitative PCR (real time PCR) Kristin Elwin Cryptosporidium Reference Unit, Wales, UK

2 Two part presentation: Introduction to real time PCR: comparison with conventional PCR detection methods / chemistries Optimisation Publishing your own assays commercial approaches, kits (e.g Primer design / Ceeram..), instruments, assay design services from companies

4 Our experiences of developing real time assays: for Cryptosporidium typing (from reading a paper to modifying, optimisation, dealing with unexpected results (cross reactions) and adaptation to create a routine ISO accredited test; and modification of others approaches (published methods) for Giardia duodenalis detection and typing.

5 Polymerase Chain Reaction: High temperature denaturation of double-stranded DNA Specific primers anneal to region of interest in a 5-3 direction: sequence at 3 end of the primer and temperature are important here (Tm primer) Thermo Fisher Scientific Extension, Taq DNA polymerase extends at optimum 72 C

6 Polymerase Chain Reaction: Optimal conditions: concentrations of Mg 2+, dntps and primers should be optimised. Buffers usually proprietary and provided with your polymerase (choose Taq carefully), others such as BSA (non-acetylated) can be added. Template concentration, annealing temperatures and cycle number. Detection of PCR products (amplicons): gel or capillary electrophoresis to determine the outcome of the end point PCR, semi quantitative at best; if properly optimised (especially cycle number).

7 Real time PCR: In principle, not a whole lot different to conventional PCR, with a few important exceptions: Conventional PCR is usually followed by another step to confirm the identity of, or characterise the amplicon using RFLP or sequencing. Real time PCR is often designed (using molecular probes) to detect and characterise in the same assay Real time PCR instruments have an excitation light source (lamp or laser) and a fluorescence detection system. Detection in real time (fluorescent read-out) a window into the reaction: all methods irrespective of chemistry link amplification with generation of fluorescence Good sensitivity and specificity Reproducible Low contamination risk closed tube system; no post-pcr manipulation Reduced hands-on time

8 Real time PCR: Faster thermocycling two step (denaturation and annealing, with extension of short amplicons achieved in between) less need for gels Reaction efficiency can be calculated, and therefore: Can be truly quantitative Suitable for all targets? length, polymorphisms, highly variable over time (e.g. flu, horizon scanning for variants) Suitable for all settings? cost, expertise, facilities

9 Detecting your PCR products All PCR pretty much occurs in the same way, primers, probe of some kind, DNA polymerase enzyme (usually Taq polymerase) dntp s, magnesium, buffer and so on. But how to detect and identify them??

Sequencing the amplicon is also useful to check specificity.

10 Detecting your PCR products It s a good start when developing assays to use gel electrophoresis just to see if your amplicon is present. Sequencing the amplicon is also useful to check specificity. DNA interchelating dye e.g. SYBR Green, EvaGreen IDT Molecular probes which bind to DNA regions between the primers

11 e.g. SYBR green DNA chelating dyes such as SYBR green change structure when bound to the minor groove of double stranded DNA (ds-dna). DNA associated (bound) dye has a stronger signal than unbound dye; therefore fluorescence increases with amplification. But non-target DNA is also detected. Primer dimers are also detected and contribute to the fluorescence.

e.g. SYBR green How to use this to differentiate between target and non-target fluorescence? - melt curve analysis At the end of thermocycling the PCR instrument is programmed to produce a melt curve.

12 e.g. SYBR green How to use this to differentiate between target and non-target fluorescence? - melt curve analysis At the end of thermocycling the PCR instrument is programmed to produce a melt curve. Starting at a temperature just above the primer Tm, it increases the temperature incrementally (e.g C), measuring the fluorescence. As the ds-dna denatures the SYBR green dissociates and the fluorescence reduces. - Melt curve analysis based on sequence-specific thermal dissociation of the dye improves certainty as a specific melt point (Tm) can be expected for a given target. Non-specific target and primer dimers dissociate at a lower temperature. High resolution melt applications e.g. RotorgeneQ, look at thermal dissociation in more detail and can (reportedly) detect an SNP.

13 e.g. SYBR green Advantages: Cheapish, intervening sequences not known, type by melt curve analysis Disadvantages: Can be subjective, not so easy to transfer technology between platforms. An indicator rather than a diagnosis.

Sequence based detection (probes) All sequence-based probe assays work on the principle that a fluorophore-labelled oligonucleotide will release detectable energy (fluorescence) when amplification

Reporters absorb energy from the instrument light source and rise to an excited state, returning to ground state on emission of this energy (as light).

14 Sequence based detection (probes) All sequence-based probe assays work on the principle that a fluorophore-labelled oligonucleotide will release detectable energy (fluorescence) when amplification occurs. Fluorophores are usually either donor / reporter or acceptor/ quencher. Reporters absorb energy from the instrument light source and rise to an excited state, returning to ground state on emission of this energy (as light). The emitted light has lower energy and longer wavelength than the absorbed light and can be transferred to the quencher, if in close proximity. Examples of reporter fluorophores include FAM, VIC, JOE, CY5 This transfer of excited-state energy is called FRET (Fluorescence Resonance Energy Transfer). When the reporter and quencher are not in close proximity as a result of template extension / 5-3 exonuclease activity, then measureable fluorescence is released. Very simply, all real time PCR assay approaches work in this manner, but the structure, design and mechanism of action changes subtly.. Excitation light from PCR instrument R Q R Q Fluorescence detected by instrument R FRET Q R Q

15 Assay chemistry Assays and the functional oligonucleotides are designed in a number of ways and can be broadly categorised and described as follows: Primer-probes e.g. hairpin and scorpion primer-probes: oligonucleotides which incorporate a primer and probe in a single looped-molecule. When the new strand of DNA is synthesised by extension of the primer region, the loop unfolds to allow the probe to bind to the new sequence and this separates the reporter from the quencher. Advantages: The loop prevents formation of primer- dimers and non-specific amplification. Fluorescent signals are stronger than other probe types. Unimolecular therefore potentially more specific. Disadvantages: can be complicated to design. Navarro et al Clinica Chimica Acta 439:

16 Assay chemistry Probes e.g. hydrolysis (Taqman): Mechanism of action relies on 5 3 exonuclease action of the DNA polymerase which during extension removes (degrades) the reporter on the template-bound probe, separating it from the quencher; releasing fluorescence from the reporter. The probe annealing temperature must be higher than primer so that it only cleaves during extension (which is at a lower temperature) rather than due to temperature change Navarro et al Clinica Chimica Acta 439:

17 Assay chemistry Probes e.g. hybridisation (Molecular beacon): molecular beacons have a single stranded loop which when in closed form keeps the reporter and quencher s in close proximity. During annealing the loops unfolds emitting fluorescence. If the template is not complementary to the probe then there will be no emission as the hairpin loop prevails over the hybridisation. Navarro et al Clinica Chimica Acta 439:

18 Assay optimisation: 1. Primers / probe design Select the target region (gene or part of,) of biological interest Align multiple sequences within genus /species to check for polymorphic regions (good and bad) Ask an online tool such as Primer BLAST for help in designing primers Or design your own primers (useful if you have the best database of sequences), but then you need to check specificity in silico, using Primer BLAST (NCBI GenBank)

19 PRIMER BLAST (NCBI) design tool input

20 PRIMER BLAST (NCBI) design tool output

21 PRIMER BLAST (NCBI) primer check input

22 PRIMER BLAST (NCBI) primer check output Top hits reassuringly are Cryptosporidium spp. Scroll down through 5000 hits and other apicomplexan genera appear Goussia, Paraschneideria Ascogregarina,Hepatozoon There are >20,000 Cryptosporidium sequences on GB (different loci)

Select conserved regions for primers and probes; published assays are a good start, but always check the following: Check suitability for task (assay design type) particularly

23 Select conserved regions for primers and probes; published assays are a good start, but always check the following: Check suitability for task (assay design type) particularly thermal considerations e.g. Primer Express (Thermo Fisher Scientific); Tm of primers should be about the same as each other, the probe at least 10 C higher than that (TaqMan assays)

24 Assay design (Taqman MGB probe): Amplicon length: Maximum amplicon size should not exceed 400 bp (ideally bases) PRIMERS Avoid primer-probe-dimer interactions, hair pins, self (pair) complementary seq etc Primers bp GC content 30-80%, there should be more Cs than Gs, Avoid repeats of Gs or Cs longer than 4 bases Avoid the last 5 nt (3 ) containing GG, CC or GC Avoid 3 T residue PROBES Avoid primer-probe-dimer interactions, hair pins, self (pair) complementary seq etc probes bp Avoid a G at the 5' end of probe (quenches fluorophore) Avoid 3 G residues especially GGG-MGB Avoid repeats of Gs or Cs longer than 4 bases Avoid runs of 6 A residues Avoid CC motif mid-probe Avoid a G residue at nt #2 at 5 end of a FAM labelled probe

Assay optimisation: 2. Reaction components Primer and probe concentration (titration, chequerboard): it is often optimal to have different primer concentrations : e.

25 Assay optimisation: 2. Reaction components Primer and probe concentration (titration, chequerboard): it is often optimal to have different primer concentrations : e.g: forward 300 nm and reverse 900 nm Then optimise the concentration of probe, using the lowest possible concentration (cheaper) F / R primer (nm) Thermo cycling conditions e.g: (95 C and 60 C, 50 cycles) (we favour a standard approach for all assays allowing multiple assay runs on the same instrument)

26 Assay optimisation: Master mix try what you have on site, what others use. Select by application: Environmental master mixes, sensi-mix etc ask for free samples from suppliers Template amount / total reaction volume 1-3 µl DNA in a µl reaction Initial checks using AGE before introducing your expensive probe run the products on a gel (as the gold standard) just to make sure you have amplicons of the correct size. Introduce the probe to make sure your conditions are good, if no fluorescence run on a gel again just to check

guidelines (Minimum Information for publication of Quantitative real time PCR Experiments).

27 Publishing new assays New assays which are useful to others should be published However, inconsistent real-time PCR experiments which cannot be reproduced have recently been a problem leading to the development of the MIQE guidelines (Minimum Information for publication of Quantitative real time PCR Experiments). Essential and Desirable items are listed, reviewers and editors are strongly encouraged to apply these. Part 2 description of trying to do this!!

28 Instrument choice An open platform, accessible to all (most) chemistries is useful e.g. RotorgeneQ (Qiagen), Cost What else might you want it for, how many fluorescent channels? Try before you buy Already available in your department

29 Commercial kits and assay design Numerous kits available for detection of some FBP using real-time PCR (e.g. Primer Design, Ceeram, Fast Track diagnostics) Suitability for your matrix Target gene (you might choose a different one) Detection / characterisation (some FBP this is important) Some companies offer design services Cost? Post development modification IP and publication rights

30 back in 10 minutes

31 CRU Cryptosporidium and Giardia assays What did we need to do? Cryptosporidium: Testing samples per year (PCR-RFLP laborious, COWP not discriminatory enough) For the purpose of molecular epidemiological surveillance Stream line workflow (target most common species, deal with the others later ) Embrace new technology (real-time PCR) Reduce waste and exposure to chemicals (gels / DNA dyes) Giardia: New workstream typing in Wales For the purpose of molecular epidemiological surveillance Immediate use of new technology Stream line workflow (target most common assemblages) Further projects detection and typing all assemblages

Cryptosporidium typing from stools Literature review of published methods (always worth checking!) Sultan Tanriverdi (2003), (Tufts University, Boston) published C. hominis and C.

32 Cryptosporidium typing from stools Literature review of published methods (always worth checking!) Sultan Tanriverdi (2003), (Tufts University, Boston) published C. hominis and C. parvum specific multiplex assay (based on melt curve analysis of Lib13 locus) Redesigned primers and developed probes creating a TaqmanMGB assay Shared forward primer and species-specific reverse primer and probe Original primers new primers and probes

horse genotype: (easily recognisable, ID by 18s sequencing) 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.

33 Norm. Fluoro. Cryptosporidium typing from stools Optimised and rolled out in 2010: BUT: Co-amplification of C. sp. horse genotype: (easily recognisable, ID by 18s sequencing) Threshold Cycle Co-amplification of C. hominis and emerging C. cuniculus (indistinguishable from each other more of a problem)

34 Cryptosporidium typing from stools Replaced C. hominis Lib13 with A135 locus primers and probe (adapted from Fabio Tosini, 2010) to remove cross reactions with C. cuniculus. C. viatorum cross reactivity further adaptation of A135 (new spp. gotta keep up with emerging species!!) alignment alignment alignment. Finally have satisfaction

35 Cryptosporidium typing from stools But, in silico predictions are very useful but NOTHING replaces actually trying your assay out on real DNA. C. cuniculus cross reactions, easily identifiable and confirmed by 18s sequencing

Demonstrate sensitivity e.g. using counted organisms (gdna) or plasmid DNA: C. parvum Lib13 assay: gdna 48 copies / PCR (3/3), 4.

36 Analytical specificity and sensitivity After in silico design and optimisation on intended targets Need to test on non-target species (same genus) e.g. C. canis. C. meleagridis, C. ubiquitum etc, and likely present non-target species (different genera) e.g. Giardia, Cyclospora, Microsporidia, Human DNA, yeasts. Demonstrate sensitivity e.g. using counted organisms (gdna) or plasmid DNA: C. parvum Lib13 assay: gdna 48 copies / PCR (3/3), 4.8 copies / PCR (1/3) pdna 3 copies / PCR (5/5), 0.3 copies / PCR (2/5) (greater enumeration accuracy)

37 Diagnostic specificity and sensitivity C. parvum assay specificity: 110 true negative stools and 49 true positive stools = 1.00 (95% CI 0.93 to 1.00) C. parvum assay sensitivity: 110 true negative stools and 49 true positive stools = 1.00 (95% CI 0.97 to 1.00) C. hominis assay specificity 110 true negative stools and 47 true positive stools = 1.00 (95% CI 0.97 to 1.00) C. hominis assay sensitivity 110 true negative stools and 47 true positive stools = 1.00 (95% CI 0.92 to 1.00) Positive stools from the UK national collection of oocysts and negatives from the local diagnostic laboratory both pre-tested using nested Cryptosporidium 18s PCR.

38 Repeatability and reproducibility Repeatability and accuracy: One low sample (100 oocysts / µl) and one high (1000 oocysts / µl) tested 20 times each. C. parvum low mean 100.8, high mean C. hominis low mean 104.3, high mean Reproducibility: 20 samples tested on different days by three different scientists: C. parvum variance homogenous with 95% confidence p=0.969 C. hominis variance homogenous with 95% confidence p=0.923

39 Evaluation of the reaction dynamics qpcr PCR efficiency: theoretical maximum of 100% assumes that from the calibration (standard) curve that the amount of product doubles each cycle (90-105% acceptable). Linearity: correlation coefficient R 2 indicates that the concentration of an unknown can be determined from the standard curve (>0.98 acceptable)

41 Validation and accreditation ISO (clinical laboratory) ISO (FWE laboratory) Assessment of technical competence which includes (amongst a lot of other measures) proper validation of assays.

43 Creating a testing algorithm The C. hominis and C. parvum species specific real time PCR assay detects and characterises ~98% of our samples with about 50% of these C. hominis and 50% C. parvum The others are tested using conventional nested PCR (SSU rrna gene) followed by Sanger sequencing Important to know your assay s limitations and be prepared to create a testing stream which detects what you need it to CRU laboratory testing (per year) Around 5000 human stool samples animal faecal samples Drinking water related samples Milk! Lettuce and raspberries (R&D)

44 Giardia duodenalis typing from stools Assemblages A and B New testing workflow was required for Giardia-positive stools (diagnosed by microscopy) Started out by replicating published assay (Almeida 2010) using a SYBR Green and melt-curve analysis detection Originally described for the Light-Cycler, we tried it on our Rotorgene.

45 SIMPLEX ASSEMBLAGE A PCR

46 SIMPLEX ASSEMBLAGE B PCR

47 DUPLEX ASSEMBLAGE A&B PCR

48 Duplex MCA appears to show cross reactions and time was running short Real time PCR (Taqman assay) needed Typing of assemblages A&B only

49 Taqman probe-based assay development Original primers (MCA): assemblage A assay Original primers (MCA): assemblage B assay

50 Taqman probe-based assay development Modified primers and added probe, assemblage A assay: Modified primers and added probe, assemblage B assay: Modifications to length and composition to improve Tm

51 Assay optimisations / evaluation Multiplex assay (separate A&B assays in a single tube), initially set up as singleplex: Cross talk with FAM fluorophore being detected in the yellow channel from tubes where no VIC labelled probe present

52 Assay optimisations / evaluation Multiplex assay No cross talk where VIC labelled probe present

53 Assay evaluation: analytical sensitivity: A assay

54 CRU laboratory testing (project 2012.) 509 human stool samples 75% assemblage B (n=370), 7 cases with A&B 18 (~4%) cases where Giardia assemblages A and B were not confirmed or detected, but microscopy-positive for Giardia So, we need a pan genus assay for detection of all species and assemblages

Giardia typing from stools pan genus assay Literature review, to look for what is already out there Mix and match try one primer from one publication and another from somewhere else to achieve

55 Giardia typing from stools pan genus assay Literature review, to look for what is already out there Mix and match try one primer from one publication and another from somewhere else to achieve the size and sequence you need Draw a gene map very useful for visualising which regions have already been looked at (and where sequence data is likely available from) understanding the geography!

56 The Cryptosporidium 18S gene with corresponding primer sites and major hypervariable regions. Morgan et al. (1997) Johnson et al. (1995) & Nichols et al. (2003) & DiGiovanni et al. (WRF 4284) Ryan et al. (2003) Xiao et al. (2001) & Jiang s modified reverse (2005) Hadfield et al. (2011) Coupe et al. (2005) Hashimoto et al. (2006) Mary et al. (2013) CRU STN18S (under development) (bp) Xiao et al. (1999) - Hypervariable region (these regions contain a large amount of polymorphisms between species, but it must be remembered that there are also other areas containing a few polymorphisms that could also be diagnostic as well as changing RFLP cut-sites) - TaqMan probe

57 Literature review check scribble, make notes!!

58 Beta-Giardin pan-genus assay Align the chosen primers with published sequences (assemblages A-G) Design probe

59 Does it work??

A & B based on tpi gene Twin duplex reactions: incorporating (1) Giardia spp.

60 Final Giardia workflow qpcr for detection and quantification of Giardia genus based on beta giardin gene alongside a PCR for detection of human pathogenic Giardia duodenalis assemblages A & B based on tpi gene Twin duplex reactions: incorporating (1) Giardia spp. detection, inhibitor recognition and (2) assemblages A and B identification IC : Cy5 BG: VIC Tpi B : FAM TpiA : VIC

61 Summary: Real-time and qpcr development and validation Development Assay design (target sequence-based) Optimisation (thermodynamics & chemistry) Appraisal (linearity & efficiency) Initial analytical specificity Validation Analytical sensitivity Analytical specificity Diagnostic sensitivity Diagnostic specificity Precision (Repeatability & Reproducibility) Accuracy (qpcr)

62 Thank you! Any questions now or anytime this week?

Module1TheBasicsofRealTimePCR Monday, March 19, 2007

Module1TheBasicsofRealTimePCR Monday, March 19, 2007 Objectives Slide notes: Page 1 of 41 Module 1: The Basics Of Real Time PCR Slide notes: Module 1: The Basics of real time PCR Page 2 of 41 Polymerase Chain Reaction Slide notes: Here is a review of PCR,