Transferring assays between different platforms

Transferring assays between different platforms
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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