Workshop Real time PCR 12. & 13. February Tutorial 1: PCR Real Time PCR FTD Kits (FTD Respiratory PLUS)

Transcription

1 Workshop Real time PCR 12. & 13. February 2011 Tutorial 1: PCR Real Time PCR FTD Kits (FTD Respiratory PLUS)

2 Principle of PCR Template (DNA), which should be copied 1. Denaturation Primer= sequence specific nucleotides Enzym Polymerase 2. Hybridisation Nukleotids Buffer 3. Elongation

3 Principle of PCR 3 2 Endpointanalysis 1 1.Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive 2. Linearity stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dntps and primers causes them to become limiting. 3. Plateau: No more product accumulates due to exhaustion of reagents and enzyme

4 Classical PCR Amplification and detection are two independent steps Amplification => thermocycler Detection => Gel Agarosis - gel electrophoresis PCR 3-4 hours UV visualisation

5 Real time PCR

6 Real time PCR is... The ability to monitor or visualize accumulation of PCR products using fluorescence Amplification and detection are combined in one analytical step Amplification => thermocycler Detection => thermocycler Amplification Visualization < 2 Stunden

7 Why Real-Time PCR? Rapid cycling times (1,5 hour) High sample throughput (~200 samples/day) Low contamination risk (sealed reactions) Very sensitive (3pg or 1 genome eq of DNA) Broad dynamic range ( copies) Reproducible (CV < 2.0 %) Allows quantitation of results Software driven operation

8 Real Time PCR is the ability to monitor or visualize accumulation of PCR products using fluorescence Real-time Requirements 1.Reporter : A fluorescent reporter where increase in fluorescence must be proportional to the increased amount of DNA (amplicon) 2.Instrument to excite & detect reporter and be able to perform analysis 3.Analysis : Method to discriminate positive and negative results and quantification.

9 PCR equipment...a few machines Rotorgene chanels ABI Chanels Rotorgene Chanels

10 Reporter methods Intercalating Dyes : SYBR Green I Hybridization Probes Hydrolyzable probes : Taqman Displaceable probes : Molecular Beacon FRET probes : LC640, LC705 Labeled Primers : Amplifluor primers Mix primer/probe : Scorpions TM

11 Intercalating Dyes : SYBR Green I Easy Use with existing PCR primers Non-specific Detects all double stranded products, including primer dimers Need to optimize carefully so no nonspecific amplification OK for presence / absence detection but not great for quantitative measurements Problem - Primer Dimer Artefact:Non-sequence specific fluorescent intercalating agent quantify all double stand DNA including non-specific amplification and primer-dimer complex - Another problem is that longer amplicons create a stronger signal (if combined with other factors) this may cause saturation

12 Taqman The probe is labbeled with: R= Reporter Q= Quenscher

13 Taqman The probe is labbeled with: R= Reporter= Fluorophore molecule that emits light of a certain wavelength after having absorbed light of a specific, but different wavelength first the emission wavelength is always higher than the absorption wavelength a lot of different fluorophores are available: FAM, VIC, Hex, Ned, Rox, Cy5... Q= Quencher molecule that accepts energy from a fluorophore in the form of light and dissipates this energy either in the form of light or heat different fluorophores are available: MGB, BBQ, Tamra...

14 The probe with reporter and quencher anneal with the target DNA-singlestrand The probe is intact, there is no signal Taqman Then the primer bind and elongate the strand The labeled probe is seperated and demaged

15 The polymerase then carries out the extension of the primer and replicates the template to which the TaqMan is bound. Taqman The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule away from the close vicinity of the quencher. The fluorescence intensity of the reporter dye, as a result increases. This process repeats in every cycle and does not interfere with the accumulation of PCR product.

16 Multiplexing

17 Multiplex-PCR is a rapid method of detecting multiple targets in a single reaction PCR reagent mix contains multiple sets of primers and probes. Any one of these may be amplified during the PCR Primer/probe sets to multiple organisms Primer/probe sets to multiple target genes in the same organism Major advantage is the reduction in test processing time

18 FTD FLU Multiplex PCR detects and differentiates in one tube: Influenza A Influenza B A(H1N1) swl Internal Control

19 Real time PCR-Multiplexing

20 Fast-track Diagnostics

21 FTD/Glasgow synergies Glasgow designs tests, uses them routinely, finds out what doesn t work so well, re-evaluates Transfers to FTD FTD converts into kit format, does intra- inter-test variability, evaluates on multiple commercial machines etc and embeds in quality system FTD CE marks Glasgow updates FTD as changes introduced

22 Widest range of clinically important menus Once you know how to do one test, you can do them all Same extraction protocol One cycler program for all tests

23 September 2010: 6 tube multiplex PCR for detection of 25 respiratory pathogens 1. Multiplex: Influenza A, Influenza B, Rhinovirus (detecting also new subtype C) and A(H1N1)swine 2. Multiplex: Coronavirus NL 63, 229E, OC43, HKU 3. Multiplex: Parainfluenza 2, 3, 4, Internal control 4. Multiplex: Parainfluenza 1, Human metapneumovirus A and B, Bocavirus, Mycoplasma pneumoniae 5. Multiplex: Respiratory syncytial viruses A and B, adenovirus, parechovirus, enterovirus 6. Multiplex: Chlamydia pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae B, Staphylococcus aureus

24 - Moraxella catarrhalis - Haemophilus all types - Klebsiella - Bordetella pertussis - Legionella - Pneumocystis jiroveci

25 FTD Respiratory PLUS Color Label Contents Tubes for 12 reactions Yellow Flu Rhino PP Primer/probe mix for Influenza A, B; H1N1 and Rhino Red COR PP Primer/probe mix for Corona 43, 229, 63 and HKU Blue Para 2/3/4 Primer/probe mix for Para2, 3, 4 and IC 8 x 8 x 8 x Purple Bo-MP-Pf1 PP Primer/probe mix for Boca, HMPV A&B, M. pneumoniae and Para1 Green RsEPA Primer/probe mix for RSV A&B, entero, parecho and adeno Yellow RespBac PP Primer/probe mix for S.aureus, S.pneumococcus, HiB, C. pneumoniae Red Resp PC Postive control pool for FluRhino PP, COR PP, Para2/3/4 PP, Bo-MP-Pf1 PP and RsEPA PP Red Resp Bac PC Positive control pool for RespBac PP 8 x Blue IC Internal control 8 x Black NC Negative control 8 x 8 x 8 x 8 x 8 x

26 THE CONTROLS The three required controls are included in the kit Positive control is required to check if the application works i.e. if the pathogen is detected by the reagents. (FTD : plasmid pools for all pathogens) Negative control is important to prove that the application provides negative results if no pathogen is present in the sample (wrong positive results or false positives ). (FTD : negative control is extractable) Internal control checks for wrong negative results or false negatives. This may be due to inhibition by extraneous substances present in stool or due to human error. (FTD: RNA or DNA viruses from plant or animals in denaturant agent) All three controls are of prime importance and only if all three control reactions work properly, you can be sure that your results are reliable.

27 Process :Documentation

28 Process :THE RAW MATERIAL Swabs, tissue, cells, blood, saliva. depends on the pathogen, that you want detect This test is for use with extracted RNA and DNA from respiratory samples (throat/nasal swabs, bronchoalveolare lavage, sputum) of human origin.

29 Enzyme Mastermix room Main lab PCR machines room Extraction room Process: Extraction 1. Thaw one negative control (NC) and one internal control (IC) 2. Extract 12 samples and the NC. We recommend a starting volume for the extraction of 300 ul and an elution volume of 75 ul. 3. Add 3 ul internal control (IC) directly to the lysis buffer of each extraction 4. Do not extract positive controls

30 Enzyme Mastermix room Main lab Extraction room Process: Preparation of the PP mixes PCR machines room 1. Thaw reagents for the reaction: Flu/Rhino PP, COR PP, Para2/3/4 PP, Bo/Mp/Pf1 PP, RS/EPA PP, RespBac PP, PC, RespBacPC and also the enzyme and the mastermix of AgPath-ID One-Step RT-PCR Kit 2. Pipette the required amount of the 2xRT PCR buffer and 25x RT-PCR enzyme mix (AgPath-ID One-Step RT- PCR Kit) to the PP mixes

31 Enzyme Mastermix room Main lab PCR machines room Extraction room Process: Preparation of the plate 1. Pipette 15 Pl of each PP mix with the 2xRT PCR buffer and the 25x RT-PCR enzyme mix in the wells 2. Add 10 Pl of the extracted samples, the extracted negative control and the positive control

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33 Universal thermal cycling protocol Depends on the enzyme that you use Reverse Transcriptase Activation of the Polymerase Depends on the primer and probes that you use Denaturation Annealing

34 Setting of the thermocycler

35 Thank you.

36 Stool: viruses, parasites and bacteria 3 kits one sample Pathogens Two tube mulitplex: norovirus G1/G2, astrovirus, rotavirus and adenovirus One tube multiplex: entamoeba histolytica, cryptosporidium sp. and giardia sp. Two tube multiplex: Shigella ssp. (EIEC), Yersinia enterocolitica, Campylobacter jejuni, C.coli, E.coli (EHEC including Verotoxin 1 or 2), Salmonella enterica, Clostridium difficile (Toxin B)

37 FTD Bacterial gastroenteritis Tested against a range of salmonella species species positive FTD negative total salm culture In-house positive 56 / negative / shig culture In-house positive 4 / negative 1* a cdiff culture In-house positive 27 / negative 2* b yers culture In-house positive 6 / negative / campy culture In-house positive 113 / negative 1* c EHEC vtx+ culture In-house positive 15 / negative 2* d