RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and Microbial Identification w/ Excellent Efficiency, Reproducibility, and Support
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The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and Microbial Identification w/ Excellent Efficiency, Reproducibility, and Support
- 1. Sample to Insight RotorGene Q – A Rapid, Automatable real-time PCR Instrument for Genotyping and Microbial Identification w/ Excellent efficiency, Reproducibility, and Support James Qin Senior Scientist, MDx Applications, Qiagen Webinar-related questions: QIAwebinars@QIAGEN.com 1
- 2. Sample to Insight Legal disclaimer 2 QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor. Pathogen detection with HRM and multiplex RT-qPCR
- 3. Sample to Insight 4 Agenda 3 Introduction to Rotor-Gene Q Unique real-time PCR chemistries and kit portfolios QIAglity - An efficient, sensitive liquid handler for real- time PCR setup Applications: • Sensitive target measurement in quantitation • Good multiplex-PCR with no inhibition and cross- talk • Quantitative high-resolution melting (HRM) in genotyping and microbial ID 1 2 3
- 4. Sample to Insight 4August 2013 Thermal cyclers (block): Peltier Technology Characters of a “Traditional” Real-time PCR Instrument Thermal precision (uniformity) in a block-based cycler + 0.50 ºC variance across the 96 well block (1.0 oC difference) Corner and edge wells most affected (fluorescence 1/temperature) Can cause localized “hotspots” at Peltier device junctions Hot spots can reduce Taq polymerase activity Can adversely affect QPCR data
- 5. Sample to Insight Title, Location, Date 5 Reaction Chamber PMT Detector Assembly LED Light Source Assembly Tubes in Rotor Spin Past Optics Lens Detection Filters Spindle/Motor Assembly Introduction to Rotor-Gene Q Unique design for thermal and optical precision Cross-section view (front)
- 6. Sample to Insight Title, Location, Date 6 Cool air in Centrifugal fan Drives air into chamber Centrifugal fan drives air around chamber Chamber vent opens expelling hot air Heater elements switch off Note: holes in the rotor allow free airflow Rapid air exchange for fast heating and cooling Temperature uniformity from well-to-well is + 0.02oC Cross-section view (side)
- 7. Sample to Insight 7 The Rotor-Gene Q: Optics High power light-emitting diodes (LED): • Highly robust LED technology (Mean time between service visits >50 months) • Constant and consistent output from run to run (no decay). • Uniform light-path to well (all wells excited at same intensity), so no optical normalization and no reference dye (ROX) required. • Different wavelengths to optimally excite fluorophors at abs max • LEDs are highly stable with lifetime guarantee (no lasers or lamps to replace) Sensitive PMT (photomultiplier tube) detector Excitation source: LED Rotor spins tubes at 400 rpm Filter set (rotates for each channel) Lens
- 8. Sample to Insight Title, Location, Date 8 Excitation (LEDs) Detection (Filters) Supports all QPCR detection chemistries: Intercalating dyes (eg. SYBR Green) Hydrolysis probes (TaqMan) Hybridization probes (FRET) Up to 6 individual channels with wavelengths spanning UV to infra-red. Ideal for multiplexing Unmatched versatility in dyes and channels Rotor-Gene Q Optical Range
- 9. Sample to Insight Title, Location, Date 9 Rotor-Gene Q accessories Easy to set-up! Rotors are interchangeable and snap-in like a DVD. Locking ring secures tubes in place during the run Option to use 0.2 mL tubes or 0.1 mL microtubes (strips of 4) or Rotor-Disc (100-well) RGQ includes accessories pack with multiple rotors, setup blocks, rotor-holder and plasticware starter set. 1 year warranty
- 10. Sample to Insight 10 *OTV disc The OTV system* uses the chemical properties of three different thermochromic liquid crystals (TLCs) as an absolute temperature reference. TLCs change from opaque to transparent at a very precise temperature s (transition point) of 50, 75 and 90oC The reported value is compared to the known calibrated value to verify the instrument is within specification. If not within specification, automatic re-calibration of the Rotor- Gene can be done at the press of a button. *patent pending Easy, routine verification of in-tube temperature in 30 minutes! Temperature verification on the Rotor-Gene Q:
- 11. Sample to Insight 11 Rotor-Gene Q Instrument Options To meet your specific laboratory requirements: Rotor-Gene Q 2plex 2 channel intro offer with highest performance Rotor-Gene Q 2plex HRM a cost effective entry point to QPCR & HRM Rotor-Gene Q 5plex full flexibility for all qPCR applications Rotor-Gene Q 5plex HRM maximum versatility in your research Rotor-Gene Q 6plex unmatched multiplexing possibilities All packages include: Real time PCR System & laptop General accessories & plasticware starter set 1 year warranty 1.5 day installation & training (optional)
- 12. Sample to Insight 4 Agenda Pathogen detection with HRM and multiplex RT-qPCR 12 Introduction to Rotor-Gene Q Unique real-time PCR chemistries and kit portfolios QIAglity - An efficient, sensitive liquid handler for real- time PCR setup Applications: • Sensitive target measurement in quantitation • Good multiplex-PCR with no inhibition and cross- talk • Quantitative high-resolution melting (HRM) in genotyping and microbial ID Applications 1 2 3
- 13. Sample to Insight 13 QIAGEN Sample to Insight solutions for pathogen and microbial extraction Detection and analysis • QIAmp DNA Mini Kit • QIAmp UCP Pathogen Mini Kit • QIAmp DNA Stool Fast/ Mini Kit • QIAamp DNA Microbiome Kit • QIAamp 96 DNA QIAcube HT Kit • QIAsymphony mericon Bacteria Kit MO BIO Laboratories for NGS • PowerSoil DNA Isolation Kit • PowerFecal DNA Isolation Kit • … • QuantiNova Probe PCR kits • QuantiNova RT-PCR kit • QuantiFast Pathogen +IC kits • QuantiTect Virus kits • Type-it HRM PCR kit for genotyping assays • Microbial DNA qPCR Arrays and Assay Kits • Rotor-Gene Q • QIAxcel DNA Kits • Allprotect • InhibitEx • RNAlater Pathogen detection workflow: From Sample to Insight Sample isolation Sample collection & Stabilization qPCR or qRT-PCR Pathogen detection with HRM and multiplex RT-qPCR
- 14. Sample to Insight The enzymes: ▪ HotStarTaq Plus and antibody-mediated hot-start DNA Polymerases ▪ Reactivation within 5 minutes ▪ Unmatched specificity and sensitivity The buffer: ▪ Unique combination of K+ and NH4 + ions ▪ High specificity ▪ No optimization necessary Q-BondTM: ▪ Enables fast cycling by promoting annealing of Taq and primer/probe to the template Synthetic Factor MP ▪ Supports macromolecular crowding ▪ Enables equal amplification of templates that differ in abundance Unique buffer composition – Solving problems with fast real-time PCR High PCR specificity and macromolecular crowding Pathogen detection with HRM and multiplex RT-qPCR 14
- 15. Sample to Insight 15 QIAGEN Genotyping product Type-it HRM PCR Kit ▪ Fast and accurate detection of gene mutations and SNPs by High-Resolution Melting (HRM) analysis Scan for mutation Type mutation Dye Saturation/Relocation w/ SYBRGreen II Dye (FAM) Dye Saturation/Relocation w/ SYBRGreen III Dye (EvaGreen, CYTO9)
- 16. Sample to Insight 16 QIAGEN RT-qPCR and multiplex PCR products QuantiTect Multiplex PCR Kit and QuantiTect Probe PCR Kit ▪ Real-time PCR and two-step RT-PCR Wide dynamic rangeHigh specificity Fast: Faster results with time savings of up to 50% Multiplex: Successful multiplex PCR without the need for optimization Sensitive: Detect up to 4 targets in 1 tube Reliable: Quantify low- and high-abundance targets, no optimization Pathogen detection with HRM and multiplex RT-qPCR
- 17. Sample to Insight 17 QIAGEN RT-qPCR and multiplex PCR products (2) QuantiNova Probe RT-PCR Kit and QuantiNova SYBR® Green RT-PCR Kit ▪ Novel QuantiNova two-phase hot-start mechanism Reliable: Increased reliability of gene expression results using gDNA reduction Convenient: Room-temperature reaction setup without compromising Visual pipetting control: limit pipetting errors Internal controls: positive in-process verification of successful RT-PCR and monitoring RT- PCR inhibition Sensitive: High sensitivity for low-copy RNA targets Pathogen detection with HRM and multiplex RT-qPCR
- 18. Sample to Insight 4 Agenda 18 Introduction to Rotor-Gene Q Unique real-time PCR chemistries and kit portfolios QIAglity - An efficient, sensitive liquid handler for real- time PCR setup Applications: • Sensitive target measurement in quantitation • Good multiplex-PCR with no inhibition and cross- talk • Quantitative high-resolution melting (HRM) in genotyping and microbial ID 1 2 3
- 19. Sample to Insight QIAgility Liquid Handler Overview & Comparison QIAgility QIAgility was specifically developed for PCR setup, and other liquid handling actions. Advantages Easy to use software Small footprint Programmed applications Plug & play Large Liquid Handlers Hamiltons, Beckmanns, Tecans and more…. These instruments are real liquid handlers not specifically developed for PCR setup. Disadvantages Complex to programm Huge instruments – lab space limited No programmed applications long installation Examples: Starlet, Biomek, Freedom Evo 75 19
- 20. Sample to Insight No matter what real-time instrument is used, QIAgility is compatible The system is pre-configured to work with all PCR tube types and all plate formats, including varieties of 96-well, 384-well and Rotor-Disc Up to 4 different setups can be performed in parallel Beside any PCR setup the system can perform ▪ Set-up of quantification standards (dilution series) ▪ Normalization of sample concentration ▪ Sample pooling ▪ Transfer of samples from one tube format to another ▪ Plate replication ▪ Restriction digest setup ▪ Cherry picking from archived sample banks The Hallmark of QIAgility Is Its Versatility 20
- 21. Sample to Insight QIAgility means Time Savings You have more important things to do… QIAgility performs complex set-ups faster than can be done manually Staff can be attending to other duties while the QIAgility is running Complicated runs don’t get put off because they will take too long to set up Simple Transfer Actions 1:1 96 samples ▪ 17 minutes ▪ 6 minutes with tip reuse Transfer Actions with pre-MM 96 samples PCR set-up ▪ ~25 minutes Transfer Actions with pre-MM 192 samples PCR set-up ▪ ~46 minutes 21
- 22. Sample to Insight QIAgility Applications and Protocol Support Applications Pre-written QIAGEN protocols at launch available ▪ Rotor-Gene SYBR Green PCR kit ▪ Rotor-Gene Multiplex PCR Kit ▪ QIAGEN OneStep RT-PCR kit ▪ HotStar Taq DNA Polymerase ▪ Cador BVDV RT-PCR kit / Reagent Application lab supports customized QIAGEN protocols 22
- 23. Sample to Insight RotorGene: Tests with 100-Ring Disc (100 replicates) PMT ND5A on DRE-Urine Sample 3932 (1: 10) Setup by QIAgility 81.0 ⁰C Run-1 (100 wells) Run Mean Run Stdev Run %CV TH0.10 18.69 0.0752 0.0752 Linear Curve Threshold 0.10 Log Curve Threshold 0.10 Melt Curve Threshold 0.10 Run-2 (100 wells) Run Mean Run Stdev Run %CV TH0.125 18.72 0.0802 0.0802 QIAgility PCR Setup in 100 Disc-ring reactions by RotorGene (10-ul reaction volume) 23
- 24. Sample to Insight 4 Agenda 24 Introduction to Rotor-Gene Q Unique real-time PCR chemistries and kit portfolios QIAglity - An efficient, sensitive liquid handler for real- time PCR setup Applications: • Sensitive target measurement in quantitation • Good multiplex-PCR with no inhibition and cross- talk • Quantitative high-resolution melting (HRM) in genotyping and microbial ID 1 2 3
- 25. Sample to Insight Title, Location, Date 25 Rotor-Gene Q: Applications All QPCR applications supported: Absolute Quantification (standard curve) Relative Quantification High Resolution Melt Analysis Applications: Gene expression Genotyping – allelic discrimination SNP analysis Mutation detection and screening Pathogen detection Methylation analysis STR and VNTR analysis Quantification of copy number variants and mosaicism …
- 26. Sample to Insight Bring multiple colors to your qPCR – The new QuantiNova Multiplex PCR Kit For fast real-time PCR and two-step RT-PCR with up to 6 targets Stringent antibody-mediated hot-start activation with QuantiNova Guard : Improved specificity and room-temperature setup enabling automated handling 26
- 27. Sample to Insight The latest generation in real-time multiplex PCR Built-in pipetting control for QuantiNova Multiplex PCR Kit From sample to multiplex data in 60 mins Fast results in a high multiplex format 20 min 2 min 5 s 30 s RNA template gDNA template 40 cycles Eliminate errors during reaction setup QuantiNova MP Master Mix contains an inert blue dye The sample dilution buffer contains an inert yellow dye Dyes do not interfere with the real-time PCR Ensures accurate reaction setup without impacting performance 27
- 28. Sample to Insight Gene Expression Workflow QuantiNova Reverse Transcription Kit for cDNA synthesis in 2-step qRT-PCR QuantiNova Multiplex PCR Kit for multiplex DNA or cDNA quantification RNA Stabilization and Purification cDNA Synthesis (2-step RT-PCR) q(RT)-PCR Data Analysis and Interpretation RNA templates Reverse transcription cDNA template Amplification qPCR products gDNA template Amplification qPCR products cDNA analysis — two-step RT-PCR gDNA analysis — PCR QuantiNova RT Kit QuantiNova Multiplex PCR Kit 28
- 29. Sample to Insight QuantiNova Multiplex PCR Kit - Reliable multiplex PCR from gDNA and cDNA providing unique in-process safety measures High specificity due to novel hot-start mechanism Built-in pipetting control to minimize errors during reaction setup Highest sensitivity for robust detection of rare targets Increased master mix concentration for maximal template input Multiplex capability for up to 6 targets for more results per reaction Outstanding reaction stability — ideal for high-throughput assays Compatible with all real-time PCR cyclers for more flexibility Reliable multiplex PCR offers: Higher accuracy by inclusion of Internal Control (IC RNA) or reference gene(s) More insight from limited sample material Increased workflow efficiency 29 Faster, most sensitive multiplex qPCR
- 30. Sample to Insight QuantiNova MP PCR Kit – Improvement of performance Stability immediately after rxn set-up (1) QIAGEN Roche* AB* ACTB (500) CDK1 LINE1 GAPD * LC Multiplex DNA Kit * AB TaqMan MP Kit* QN MP PCR Kit 4plex PCR reactions were set up at room temperature and run immediately after reaction setup 30
- 31. Sample to Insight QuantiNova MP PCR Kit – Stability 24 hours after rxn set-up (2) Better performance for all 4 assays with QN MP PCR Kit after 24 hours storage Reaction is stable at room temperature for 1 day after rxn setup, allowing worry-free automation QIAGEN Roche AB ACTB (500) CDK1 LINE1 GAPD 4plex PCR reactions were set up at room temperature and left at 22.5°C for 24 hours before PCR 31
- 32. Sample to Insight QuantiNova Multiplex PCR Kit – Wide Linear Range; Ideal for Single Copy applications Precise quantification of low and high abundant targets in a multiplex format: high template amount LINE1 CDK1 4 targets with varying abundance were coamplified from gDNA starting w/ 800 ng, down to 50 ng in 1:2 dilutions (Examplary of 2 targets) 32
- 33. Sample to Insight QuantiNova MP PCR Kit – Wide Linear Range (2) Precise quantification of low and high abundant targets in a multiplex format: low template amount 4 targets with varying abundance were coamplified from gDNA, starting w/ 48 ng, down to 6 pg in 1:2 dilutions Reliable quantification of changes in gene expression profiles and copy numbers over a wide range of template amount 33
- 34. Sample to Insight QN MP PCR Kit: Compatibility w/ automated setup by QIAgility vs. Manual ACTB CDK1 Cycle 5 10 15 20 25 30 35 40 45 Norm.Fluoro. 0,30 0,25 0,20 0,15 0,10 0,05 0,00 Threshold Cycle 5 10 15 20 25 30 35 40 45 Norm.Fluoro. 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 Threshold Cycle 5 10 15 20 25 30 35 40 45 Norm.Fluoro. 0,95 0,90 0,85 0,80 0,75 0,70 0,65 0,60 0,55 0,50 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 Threshold Cycle 5 10 15 20 25 30 35 40 45 Norm.Fluoro. 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 Threshold LINE1 GAPD A 4-plex PCR, using human genomic DNA (30 pg - 30 ng) was set up either manually or with QIAgility and run side by side on the same plate. Full compatibility with automated rxn setup, even after prolonged storage time at room temperature 34
- 35. Sample to Insight Unbiased Multiplex PCR Quantification of MYC in 1/2/3/4 plex PCR Unaltered quantification upon coamplification of various targets with varying abundance Template: 10 ng HeLa cDNA 35
- 36. Sample to Insight QuantiNova MP PCR Kit: Multiplex PCR of up to 6 targets Outstanding performance, multiplexing capacity and process safety 6-plex PCR on RGQ from 20 ng HeLa cDNA 18SrRNA Marina Blue LINE1 Cy5 GAPD Texas RED MYC HEX HSP90AA1 Quasar 705 TNF FAM 20ng/rxn Note: Different fluorescence intensities are caused by the use of different fluorophores in different channels Unique in-process safety measures differentiate us from competition Inclusion of IC RNA and reference gene(s) possible Visual pipetting control Excellent specificity and room temperature stability by novel hot start mechanism Efficiency and performance increase through superior multiplex format Up to 6-plex – outstanding capability to streamline experimental design – More insight from limited samples Highly concentrated enables sensitive assays design Very fast, saving time Easy to use Superior performance over existing chemistries ensuring reliable results for users 36
- 37. Sample to Insight Title, Location, Date 37 HRM characterizes double-stranded PCR products based on their detailed strand dissociation (melt) behavior: Distinguishes samples with different sequence, length, GC content Sample characterization by melt curve shape and melt temperature Many applications such as: ▪ SNP Genotyping / allelic discrimination ▪ Quantitative DNA methylation analysis ▪ Gene scanning ▪ Sequence matching A high-intensity optical HRM channel An outstanding thermal resolution of 0.02°C High data acquisition rates A comprehensive HRM software package Use with special saturating intercalating dye (EvaGreen) Mutants (Tallele) Wild types (C allele) Heterozygotes Mutants (Tallele) Wild types (C allele) Heterozygotes The Rotor-Gene Q HRM option features: The Rotor-Gene Q can detect even the most difficult class IV SNPs by HRM (with Tm difference of 0.1° C) High Resolution Melt Anlaysis (HRM)
- 38. Sample to Insight deg. 75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 100% methylated 70% 90% . 30%. 50%. 10%. 0% methylated deg. 75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 Normalisedminus50%m50%u 35 30 25 20 15 10 5 0 -5 -10 -15 -20 -25 100% methylated 70% 90% . 30%. 50%. 10%. 0% methylated Various ratios of methylated and unmethylated DNA-APC Rotor-Gene HRM + EpiTect HRM PCR Kit Standard normalized melt curve Difference plot normalized to a 50% methylated sample deg. 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0 Normalisedminuswt 15 10 5 0 Accurate SNP genotyping by HRM Rotor-Gene HRM 5plex + Type-it HRM PCR Kit + 10 ng genomic DNA Standard normalized melt curve Difference plot normalized to a wild type sample deg. 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0 NormalisedFluorescence 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 Homozygote mutant Wild Type Heterozygote Homozygote mutant Heterozygote Wild Type Genotyping with HRM on the Rotor-Gene Q is an fast and cost-effective technique with high specificity 38 Applicaation example: Epigenetic research and Genotyping w/ HRM
- 39. Sample to Insight For many datasets, most of the eigenvalues “lambda” are negligible and can be discarded. The eigenvalue measures the variation In the direction e: Example: ScreenClust S.W. for HRM and Principal Component Analysis (PCA) Finding relationship from a large multi-layer sample set is always a challenge. One way to avoid the dimensionality is by projecting the data onto a lower-dimensional space. It rotates multivariate dataset into a new configuration which is easier to interpret. Techniques for dimension reduction: Principal Component Analysis (PCA) – most effective! Fisher’s Linear Discriminant Multi-dimensional Scaling. Independent Component Analysis.
- 40. Sample to Insight Applications of PCA in Genomics Purposes… ➢ Simplify data – PCA is the most commonly used dimension reduction technique. ➢ Look at relationships between variables – PCA is useful for finding new, more informative, uncorrelated features. ➢ PCR reduces dimensionality by rejecting low variance features. ➢ Look at patterns of units – i.e. new mutations, targets ➢ Analysis of expression data ➢ Analysis of metabolomics data (Ward et al., 2003) Example Run wild type heterozygote mutant A/T Class IV SNP minute differences between homozygote alleles (< 0.1°C) 40
- 41. Sample to Insight ºC 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 dF/dT 3.75 3.50 3.25 3.00 2.75 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 JE341/Pro805 V6 V5 V3 V7 RGQ real-time PCR Melt Curves HRM: RotorGene Melt Curve Analysis on five assays for primer selection 41
- 42. Sample to Insight ºC 71.5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10 JE341/Pro805 Normalized to V5: V6 V5 V3 V7 ºC 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 0 -10 -20 -30 -40 -50 -60 Normalized to JE341/ Pro805: JE341/Pro805 V6 V3 V5 V7 Example: RotorGene HRM Analysis on designed assays for primer selection (2) HRM PCR Genotyping Kit/ RGQ – Offers Distinct Melt Curves JE341/Pr o805 V3 V5 V7 V6 42
- 43. Sample to Insight ºC 75 80 85 90 dF/dT 2.0 1.5 1.0 0.5 0.0 ºC 77 78 79 80 81 82 83 84 85 86 87 88 89 100 80 60 40 20 13003 13009 13000 13026 13031 13027 13003DS 13003DP13004 1300013029 HRM is able to pick up individual differences High Resolution Melt (V3 assay) Distinct melt curve patterns between individual subjects HRM and melt curve analysis by ScreenClust on human references in Microbiome research 43
- 44. Sample to Insight Clustering Analysis by ScreenClust on human references for potential health monitoring 44
- 45. Sample to Insight Blood Duffy antigens (FY) Genotyping HRM w. ScreenClust S.W. can be applied in blood genotyping application Kidd antigens (FY) Genotyping: PCA Analysis in ScreenClust S.W. 45
- 46. Sample to Insight Multiplex PCR products in Applied Testing applications 46 y = -3,2871x + 40,07 20 25 30 35 40 0 2 4 6 CT Log target copy number Aspergillus niger y = -3,2356x + 41,04 20 25 30 35 40 0 2 4 6 CT Log target copy number Klebsiella pneumoniae y = -3,1423x + 41,649 20 25 30 35 40 0 2 4 6 CT Log target copy number Mucor/Rhizopus spp. y = -3,1608x + 41,204 20 30 40 0 2 4 6 CT Log target copy number Pan Aspergillus/Penicillium Pathogen detection with HRM and multiplex RT-qPCR Development of real-time pathogen detection assays for food and agricultural products such as marijuana, organic drinks Extraction chemistry: QIAamp DNA kit Detection Kit: QuantiFast Pathogen+ PCR Kit Sensitivity ▪ Standard curves from 0 to 1,000,000 copies were prepared ▪ 1000 copy Ct<31 or Ct<34 (low end assays if NTC Ct=40) ▪ Primer efficiency> 80%, R>0.995, LLOQ determined
- 47. Sample to Insight T. Foetus (Fam) Internal Control (Yellow) TH = 0.05 Tritrichomonas Foetus and InType IC – A clean multiplex assay 47
- 48. Sample to Insight A clean Crypto assay in 4-plex reaction with good dynamic range Cryptosporidium in 4-plexes assay: Good consistency and sensitivity of Crypto+ in the 4-plex TH = 0.20 Calf scours panel: Ecoli K99/ Sal./Crypto/ InType 4-Plex assay 48
- 49. Sample to Insight Comparable target Cts. A good specificity on POS vs Negs; and a consistency of M. Haem in singleplex vs. 5-plex assays. Mannheimia Haemolytica in 5-plexes : Good consistency, specificity, and tighter range of M. Haem+ in the 4-plex TH = 0.05 Good signal and tight range in IC controls. InType ICM. Haem+ Bovine BRD Respiratory Panel: M. Haemolytica in 5-plexes (M. haem/H. Somni/ M.bovis/ P.mult / InType IC) 49
- 50. Sample to Insight Comparable target Cts. Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex Histophilus Somni in 5-plexes: Good consistency, specificity, and less cross-reactivity of M. Haem+ in the 4-plex TH = 0.05 A good signal and tight range in IC controls in 5-plexes. Bovine BRD Respiratory Panel: H. Somni in 5-plexes (M. haem/H. Somni/ M.bovis/ P.mult / InType 5-Plex assay) 50
- 51. Sample to Insight TH = 0.05 Better specificity and great improvement on negative samples in 5-plexes vs. H. Somni singleplex after optimization Consistent, good signal and tight range in IC controls. Pasteurella multocida in 5-plexes: Good consistency, specificity, and tighter range of P. mult + in the 5- plex Bovine BRD Respiratory Panel: P. mult + Optimization (M. haem/H. Somni/ M.bovis/ P.mult / InType 5-Plex assay) 51
- 52. Sample to Insight Questions? Thank you for attending Contact QIAGEN Technical Service Call: 1-800-426-8157 for US Call: +49 2103-29-12400 for EU Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com 52
- 53. Sample to Insight FAQs I QuantiNova Multiplex PCR Kit Is it possible to add the ROX passive reference to the master mix for long-term storage? If only Applied Biosystems cyclers will be used, QN ROX Reference Dye Solution can be added to the 4x Master Mix for long-term storage, if desired. Corresponding instructions are given in the HB. Is the QuantiNova MP PCR mix compatible with partially purified / crude samples? The kit has been developed for efficient amplification of DNA/cDNA. Typical contaminations from crude nucleic acid preparations (such as SDS or hemoglobin) will impair the results and will reduce the sensitivity and specificity of detection. What is the stability of the QuantiNova MP PCR mix at -20°C, 4°C, and room temperature, respectively? Our (preliminary) data indicate that the stability will be at least 24 months (-20°C), 12 months (4°C) and 30 days (room temperature). Is the QuantiNova MP PCR Kit compatible with degenerated primers and probes? The mix has been developed to provide highest annealing specificity to allow selective target amplification even if there are many similar target sequences. Therefore, degenerated primers and probes will not work optimally. 53
- 54. Sample to Insight FAQs II QuantiNova Multiplex PCR Kit Which reaction volumes can the QuantiNova MP PCR Kits be used with? Recommended rxn volume: 20 µl (96 well plate), 10 µl (384 well plate) Successfully tested rxn volume range: 8-25 µl (96 wells), 5-10 µl (384 wells) What is the maximal number of targets which can be coamplified? Our „official“ recommendation, as given in the QSP and the HB, are 5 targets. This is the phyisical limit of most real-time cyclers on the market. However, we have shown, that up to 6 targets can be coamplified successfully and in an unbiased manner. Which fluorophor combinations are recommended? Based on the number of different instruments on the market and fluorophore/quencher combinations offered by the oligo suppliers, there is no „gold standard“ for the optimal probe combination. Actually each instrument provider as well as any oligo supplier will provide corresponding information. On which cyclers has the QuantiNova MP PCR Kit successfully been tested? Basically almost every instrument from the major real-time PCR instrument suppliers. We did not have access to AB‘s QuantStudio instruments; however, the technology is comparable to the ViiA7 instrument, on which the QN MP PCR Kit works very efficiently. 54
- 55. Sample to Insight FAQs III QuantiNova Multiplex PCR Kit What is the minimal/maximal template input? Template amounts (gDNA as well as cDNA) up to 800 ng can be used without compromising quantification. Undiluted RT reactions can be added to the PCR reaction untill they make up 25% of the total reaction volume without impairing the quantification results. This has been tested with RT kits from QIAGEN (QuantiTect, QuantiNova) as well as with RT kits from all major suppliers (Thermo-Fisher/Invitrogen, Bio-Rad, Roche). Can the QN MP PCR Kit be combined with contamination removal using UNG (uracil-N-glycosylase)? Please note that the QN MP PCR Kit neither contains dUTP as one of the nucleotides, nor the enzyme UNG, both of which are prerequisites for an UNG treatment. However, we have very successfully tested a modified version of the kit where dTTP has been replaced by dUTP and where UNG has been added to the master mix. Amplification results were comparable to the „standard version“ and contamination removal was highly efficient (clearly above 10E6). If appropriate (revenue dependent), such a customized version can be made. Various types of probes? We have exclusively been testing TaqMan probes (both, standard TaqMan probes and MGB-containing TaqMan probes). We do not expect any problems with other probe technologies such as Hybridization probes and Molecular Beacons. Minor modifications of the protocol might be necessary (i.e. a 3-step cycling protocol with separate annealing and extensions steps). 55