Transferring assays between different platforms Topics 1. A very brief introduction to artus 2. The diversity of real-time PCR instruments 3. Incompatibility of detection format and instrument 4. PCR multiplexing complicates assay transfer instruments 5. Adaptation of a dual color pathogen detection assays (HSV and Malaria) on three different technological platforms A Very Brief Introduction to artus ü founded in 1998 as a spin-off from the Bernhard Nocht Institute (BNI) in Hamburg ü established a GMP production facility, one of the largest private owned BSE laboratories in Germany and a R&D department with extensive real-time PCR equipment (first German BSE case confirmed in 2000) ü headquarter in Hamburg, subsidiaries in San Francisco (USA) and Kuala Lumpur (Malaysia) ü focus on the development of real-time PCR based pathogen detection kits (RealArt™ kits) for several technological platforms (LightCycler®, Rotor-Gene™, ABI Prism® and SmartCycler®) (first SARS-Coronavirus detection system) The Increasing Number of Real-Time PCR Instruments LightCycler® ABI Prism® Mx4000™ Opticon®2 Rotor-Gene™ SmartCycler® iCycler Diverse Not Only in Weight... Product Company Heating Mechanism Reaction Tube max. # of Samples Weight ABI Prism ® 7000/7700/7900 Applied Biosystems Peltier Element Plates/ Tubes 96 (7900: 384) 34 kg 120 kg 82 kg iCycler IQ™ Biorad Peltier Plates/ Tubes 96/384 17.6 kg LightCycler ® Roche Diagnostics Air Capillaries 32 19.2 kg Mx4000™ Stratagene Resisitive/ Peltier hybrid Plates 96 15 kg DNA Engine Opticon ®2 MJ Research Peltier Plates/ Tubes 96 29 kg Rotor-Gene™ Corbett Research Resistive heater with air cooling Tubes 72 17 kg SmartCycler ® Cepheid I-CORE ® Tubes 16 10 kg ...but also in Temperature Uniformity... Product Max. Heating/ Cooling Rate (°C/sec) Temperature Accuracy Temp. Uniformity Volume (µl) ABI Prism ® 7000/7700/7900 1.5/1.5 +/-0.25°C +/-0.5°C up to 100 iCycler IQ™ 3.3/2.0 +/-0.3°C +/-0.4°C 10 - 200 LightCycler ® 20.0/20.0 +/-0.3°C +/-0.2°C 20 Mx4000™ 2.2/2.2 +/-0.25°C +/-0.25°C 10 - 50 DNA Engine Opticon ®2 3.0/2.0 +/-0.4°C +/-0.4°C 10 - 50 Rotor-Gene™ 2.5/2.5 +/-0.5°C +/-0.05°C 10-100 (20 rec.) SmartCycler ® 10.0/2.5 +/-0.5°C +/-0.5°C 25-100 ...and Most Importantly in Optics Product Excitation Source Excitation Wavel. (nm) Detection Wavel. (nm) ABI Prism ® 7000/7700/7900 Halogen lamp/ Argon laser 7000: 350 - 750 7000: Four filter wheel 7900: 500 - 660 7900: 488 and 545 iCycler IQ™ Halogen lamp 400 - 700 5 filter positions available (2 provided) LightCycler ® LED 470 530, 640, 710 Mx4000™ Halogen lamp 350 - 750 350 - 830 DNA Engine Opticon ®2 LED 470 - 505 523-543, 540-700 Rotor-Gene™ LED 470, 530, 585, 625 510, 555, 610, 580 hp, 610 hp, 660 hp SmartCycler ® LED 450-495, 500-550, 565-590, 630-640 510-527, 565-590, 606-650, 670-750 ABI Prism® 7900 HT ABI Prism® 7000 580 hp 610 hp Rotor-Gene™ 660 hp 510 555 610 FRET LightCycler® F1 (530) 400 500 wavelength (nm) F2 (640) 600 F3 (710) 700 Cy7 LC Red705 Cy5 Biodipy 650/665 LC Red640 ROX TAMRA VIC/JOE FAM Biodipy FL Different Instruments and Their Detection Spectra The FRET Probe Principle Works Best on the LightCycler® FRET probes are designed for the LightCycler® instrument Probe 2 LC Red640 or LC Red705 Probe 1 FAM FRET Channel Detection F1 FAM: 530 nm F2 LC Red640: 640 nm F3 LC Red 705: 710 nm Pathogen Diagnostics Requires Two PCR Reactions in One Tube Duplex PCR P quantitative analytical PCR (determination of pathogen loads) P internal control (IC) PCR - control of PCR inhibition (and extraction efficiency) - to verify negative analytical PCR results Example of a Duplex PCR Using FRET Probes on the LightCycler® analytical PCR internal control PCR LC Red640 FRET measured in F2 LC Red705 FRET measured in F3 only one excitation wavelength: 470 nm Competition Effect in a Duplex PCR HSV 1 analytical PCR in F2 IC PCR in F3 NTC NTC HSV 1 quantification standard series of defined concentrations competition between analytical and IC PCR leads to reduced fluorescence intensities Duplex PCRs in the LightCycler® Require a Color Compensation Interferences of fluorescence signals between the channels ("crosstalk") FRET LightCycler® emission spectra F1 (530) 400 500 F2 (640) 600 wavelength (nm) F3 (710) 700 Color Compensation File Subtracts Interfering Fluorescences without ccc-file NTC NTC F2 F3 with ccc-file NTC NTC 580 hp 610 hp Rotor-Gene™ 660 hp 510 555 610 FRET LightCycler® F1 (530) 400 500 F2 (640) 600 wavelength (nm) F3 (705) 700 Cy7 LC Red705 Cy5 Biodipy 650/665 LC Red640 ROX TAMRA VIC/JOE FAM Biodipy FL Transfer of the HSV Real-Time Assay to the Rotor-Gene™ Instrument Rotor-Gene™ Channel Setup Combination of different excitation and detection filters to disriminate between LC Red640 and LC Red705 on the Rotor-Gene™ excitation detection fluorophore 470 nm 610 nm LC Red640 470 nm 610 hp LC Red640/705 470 nm 660 hp LC Red705 625 nm 660 hp LC Red705 No unique channel for LC Red640 available ! No Discrimination between LC Red640 and LC Red705 exc.: 470 nm det.: 610 nm no detectable signal - emission max. of LC Red640 is higher exc.: 470 nm det.: 660 hp detection of IC (LC Red705) only exc.: 470 nm det.: 610 hp 610 hp detects all emissions of 610 nm and higher - no discrimination between LC Re640 and LC Red705 exc.: 625 nm det.: 660 hp absorption max. of LC Red705 is around 680 nm - it can, thus, not efficiently be excited Both PCR Reactions are Detected in One Channel PCR setup including IC PCR setup excluding IC exc.: 470 nm det.: 610 hp exc.: 470 nm det.: 610 hp 22.0 28.6 24.4 30.3 increase of Ct values due to overall increased detected fluorescence a LC assay cannot readily be transferred to the Rotor-Gene ABI Prism® 7900 HT calibrated with pure dyes FRET LightCycler® F1 (530) 400 500 wavelength (nm) F2 (640) 600 F3 (710) 700 Cy7 LC Red705 Cy5 Biodipy 650/665 LC Red640 ROX TAMRA VIC/JOE FAM Biodipy FL Transfer of the HSV Real-Time Assay to the ABI Prism ® 7900 Instrument Transfer of the HSV Real-Time Assay to the ABI Prism ® 7900 Instrument FAM dye layer Detection of fluorescence signals due to FAM-labeled oligo probe 1 HSV quantification standard series (101-104 copies/µl) Assay transfer Is Further Complicated by Passive Reference Dyes The ABI Prism ® instruments require a passive reference dye (usually ROX) which accounts for fluorescent fluctuations due to changes in concentration or volume in the wells. The software, thus, calculates normalized data, i.e. the ratio of reporter dye fluorescence and the emission of the passive reference (Rn = normalized reporter). The reference dye is part of the PCR Master and must, hence, be modified. ® Alternative Detection Format for Rotor-Gene™ and ABI Prism ® Instruments Use of two dual labeled probes: TaqMan probes FAM NFQ 25 - 30 nt FAM exc. max.: 494 nm det. max.: 520 nm NFQ 25 - 30 nt JOE exc. max.: 520 nm det. max.: 548 nm R analytical PCR JOE IC PCR R R: reporter fluorophore NFQ: non-fluorescent quencher Example of a Rotor-Gene™ Assay Using Dual-Labeled Probes analytical PCR in FAM channel Malaria quantification standard series of defined concentrations IC PCR in JOE channel Internal control PCR (competition effect) good fluorescence signal separation in two channels !!! No Color Compensation Required on the Rotor-Gene™ When multiplexing 4 channels, less than 1% of cross-talk is observed between channels. 580 hp 610 hp Rotor-Gene™ 660 hp 510 555 610 FRET LightCycler® F1 (530) 400 500 F2 (640) 600 wavelength (nm) F3 (705) 700 Cy7 LC Red705 Cy5 Biodipy 650/665 LC Red640 ROX TAMRA VIC/JOE FAM Biodipy FL Transfer of the Malaria Rotor-Gene™ Assay on the LightCycler® Transfer of the Malaria Rotor-Gene™ Assay on the LightCycler® F1 F2 F3 analytical Malaria PCR detected in FAM channel despite color compensation the F1 signal strikes through into the F2 channel no fluorescences can be measured in the F3 channel no IC detection as JOE cannot be excited by the LightCycler® ABI Prism® 7900 HT calibrated with pure dyes FRET LightCycler® F1 (530) 400 500 wavelength (nm) F2 (640) 600 F3 (710) 700 Cy7 LC Red705 Cy5 Biodipy 650/665 LC Red640 ROX TAMRA VIC/JOE FAM Biodipy FL Transfer of the Malaria Rotor-Gene™ Assay on the ABI Prism ® 7900 Transfer of the Malaria Rotor-Gene™ Assay on the ABI Prism ® 7900 analytical PCR FAM dye layer IC PCR JOE dye layer as ABI Prism instruments require a passive reference, the ROX dye was added to the reaction setup Other Aspects Important in Transfers of Pathogen Detection Assays ü Reaction volume may significantly affect the sensitivity of pathogen detection LC: RG/TM: reaction vol. limited to max. 20 µl allow reaction volumes of up to 100 µl increased total volume allows a larger volume of sample material ü Passive reference dyes ü Temperature profile ü ... well, try and see !
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