setting of the threshold in the middle of the linear part in a logarithmic view
DESCRIPTION
130 000 000. 13 000 000. 4200. Y(X 0 )=Y 0. 1 300 000. 130 000. 17 000. Amplification : +. 420. 42. Intermediate precision. No amplification : -. for 2, 4, 8, 10 and 16 runs data are grouped. ~ 10 calibrations (run) needed to set a robust platform cut-off value. bias. variance. - PowerPoint PPT PresentationTRANSCRIPT
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Walloon Agricultural Research Center (CRA-W)Agriculture and Natural Environment Department (D3) – Valorisation of Agricultural products (D4)
Agricultural systems, Territory and Information Technologies Unit (U11) – Authentification and traceability Unit (U16)
Léon Lacroix Building - Rue de Liroux, 9 -– Henseval Building – Chaussée de Namur, 126 - B–5030 GEMBLOUX (Belgium) Tel : + 32 (0)81 62.65.71 - Fax : + 32 (0)81 62.65.59 - Tel : + 32 (0)81 62.03.51 - Fax : + 32 (0)81 62.03.88
[email protected] - [email protected] - http://cra.wallonie.be
Setting of the threshold in the middle of the linear part in a logarithmic viewPCR Uniformity in Log Phase
-2.00E-02
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
20 22 24 26 28 30
Threshold
96 replicates
Cycle Number
Flu
oresc
ence
Ct = # cycles needed to pass threshold
Inverse regression for the determination of the cycle cut-off of a real-time PCR Inverse regression for the determination of the cycle cut-off of a real-time PCR method for the detection of bovine tissues in feedingstuffsmethod for the detection of bovine tissues in feedingstuffs
Viviane PLANCHON, Robert OGER Viviane PLANCHON, Robert OGER Aline MARIEN, Gilbert BERBEN, Olivier FUMIEREAline MARIEN, Gilbert BERBEN, Olivier FUMIERE
AGROSTAT 2010 February 23-26, 2010
Benevento, Italy
ReferencesDraper N. and Smith H. (1980) Applied regression analysis (Second edition). New York: John Wiley & Sons. Fumière O., Dubois M., Baeten V., von Holst C., Berben G. (2006). Effective PCR detection of animal species in highly processed animal by-products and compound feeds. Analytical and Bioanalytical Chemistry, 385, 1045-1054.Fumière O., Veys P., Boix A., von Holst C., Baeten V., Berben G. (2009). Methods of detection, species identification and quantification of processed animal proteins in feedingstuffs. Biotechnologie, Agronomie, Société et Environnement, 13(S), 59-70.Prado M., Berben G., Fumière O., Van Duijn G., Mensinga-Kruize J., Reaney S., Boix A., von Holst C. (2007). Detection of Ruminant Meat and Bone Meals in Animal Feed by Real-Time Polymerase Chain Reaction: Result of an Interlaboratory Study. Journal of Agricultural and Food Chemistry, 55, 7495-7501.
Exponential amplification phase
Linear scale
Logarithmic scale
Ct ~ 22 cyclesDepending of the operator:
20 < Ct < 25
Amplification : +
No amplification : -
Clear-cut signals
Cut-off
Late signals
AknowledgmentsEU Commission – DG Research and DG SANCO
RLAnimal Proteins
RLAnimal Proteins
CRL-AP : Community Reference Laboratoryfor the detection of animal proteins in feedingstuffs (2006-2011) http://www.crl.cra.wallonie.be/
0102030405060708090
100
0 1 2 3 4 5
%
Number of copies
A percentage of 95 % of blocks of triplicates detected corresponds to a level between 1 and 2 copies of the
target
ResultsDistribution of the cut-off values calculated
Percentages of blocks (of 3 replicates) detected as positive
The cut-off of the platforms are distributed on a wide range of Ct values
37.73 < cut-off < 43.68
ABCDEF
2 3 4 5 6 107 8 9
640 copies 160 copies 40 copies
1211
HG
1ABCDEF
2 3 4 5 6 107 8 9
6 calibrations
640 copies 160 copies 40 copies
1211
HG
1
PCR controls0 copy
PCR controls80 copies
Blind samples : to be analysed in triplicates : 5, 2, 1, 0.6, 0.4, 0.1
copy/PCR
Design of the inter-laboratory study
19 laboratories: 17 from the European Union, 1 from Japan, 1 from Australia
6 plates with 6 calibrations on each plate
Precision of cut-off estimation and european inter-laboratory study
Statistical aspects of inverse regression
Background
Statistical aspects of inverse regression and cut-off estimation
Application to cut-off estimation
Conclusions
Concept of threshold cycle
Real-Time PCR test
0
0.5
1
1.5
2
2.5
3
29 30 31 32 33 34 35 36 37 38 39 40 41
CP
Lo
g (
Nu
mb
er o
f co
pie
s)
640 copies/5 µl320 copies/5 µl
160 copies/5 µl80 copies/5 µl
40 copies/5 µl
10 copies/5 µl
Initially, calibrations with 28 points : 7 levels and 4 replicates/level
Based on bias, variance and practical aspectsdecision for calibrations with 9 points : 3 levels, 3 replicates/level
Evolution of the mean and the variance of XUNumber of runs to evaluate XU ?
ABI7000 ABI7500 LC480Mean 40.24 38.87 40.09
Repeatability sr 0.799 0.608 0.375r 2.21 1.69 1.04r% 5.50 4.34 2.59
CRA-W Inter thermocycler precision
ABI7000 ABI7500 LC480Mean 40.24 38.87 40.09
Reproducibility sR 1.124 0.824 0.476R 3.12 2.28 1.32R% 7.74 5.88 3.29
Intermediate precision
The cut-off determination was solved statistically using calibrations curves with plasmids carrying the PCR target and the application of inverse regression (Draper and Smith,1998) between the logarithm of the copy number and the Ct
The cut-off value is defined as the upper value (XU) of the confidence interval for the Ct values corresponding to 1 copy of the
target XU is specific of a PCR platform
X0
XY 10
Inverse regression
XbbY 10ˆ
0Y0X̂
from
?and for a
specific value
Y(X0)=Y0
known value
unknown value
Inverse regression for the determination of the cut-off for Y0=0
y = 10.914 -0.2774 x
0.5
1
1.5
2
2.5
3
28.00 30.00 32.00 34.00 36.00 38.00
CP
Lo
g (
nu
mb
er o
f co
pie
s)
Y-axis : dependant variable
predicted by X
X-axis : independant variable allow to predict Y
SAFEED-PAP : Detection of presence of species-specificprocessed animal proteins in animal feed (2006-2009)http://safeedpap.feedsafety.org/
In 1987 : bovine spongiform encephalopathy (BSE) epidemic. Most probable dissemination way of the disease = feeding with meat and bone meals (MBM)
In 2000 : TOTAL BAN on the use of processed animal proteins (PAPs) in feed decided by European Commission (EC) in order to eradicate BSE in Europe (Council decision 2000/766/EC, Regulation 2001/999/EC, 2002/1774/EC)
Progressive lift of the ban could be considered by the EC, if there is NO DANGER for the health or the policy for eradication of BSE (TSE Road Map 2005)
Need of reliable analytical methods and tools for the species-specific detection and the quantificationof PAPs in the feedingstuffs : SAFEED-PAP European project. Objective of the project : to develop and validate a suitable PCR kit for the species-specific detectionof bovine proteins in compound feed BUT the test is qualitative and requires a criterion to determineif a result is positive or negative : CUT-OFF DERTERMINATION
• PCR can sometimes give late signals that are not significant and such results have to be considered as negative. To that purpose, a cut-off value must be determined.
• It is generally expressed in terms of cycles to be compared to the Ct of an amplification curve. This Ct is the threshold cycle which means the number of cycles required for that amplification to reach a given fluorescence threshold. If the Ct of an amplification curve is higher than the cut-off, the result is considered as negative; however the Ct is a relative concept that is dependent on the thermocycler, the reagents used and the way to set the fluorescence threshold.
• At CRA-W, the cut-off was initially determined empirically to correspond to 40 cycles with the specific PCR conditions and the way the fluorescence threshold is set.
Concept of cut-off
ObjectivesTo define a scientifically sound way to find out rapidly what is the cut-off value of any other PCR platform (thermocycler, reagent, laboratory environment)
To evaluate the repeatability and inter thermocycler variability (intermediate precision) and reproducibility of the cut-off estimated through an inter-laboratory study
42
420
4200
17 0
00
130
000
1 30
0 00
0
13 0
00 0
00
130
000
000
Classical linear regression
Confidence intervals of anestimated value X0
XXUU
~ 10 calibrations (run) needed to set a robust platform cut-off value
1
2
48
10 160.00
0.04
0.08
0.12
0.16
0.20
0 4 8 12 16 20
Nb runs
Var
iance
of X
U
1
2
4 810
16
39.80
39.82
39.84
39.86
39.88
39.90
39.92
0 4 8 12 16 20
Nb runs
Mea
n o
f Xu
for 2, 4, 8, 10 and 16 runsdata are grouped
Number of copy levels and replications to evaluate XU ?
Repeatability
A scientifically sound way to find out rapidly what is the cut-off value of any other PCR platform (thermocycler, PCR reagent) has been determined based on inverse regression. Accuracy has been evaluated through evaluation of trueness and precision during an inter-laboratory study. The protocol designed for determination of the cut-off value is fit for purpose
bias variance
Real-time PCR = enzymatic reaction for the amplification of DNA fragment with a fluorescent detection. The production of fluorescence at each cycle depend on the concentration of targeted DNA in the reaction.
unknown value
known value