Real-Time PCR (qPCR),
a performing method to check
for the presence of banned
substances
Renaville R.
Progenus sa
Gembloux, Belgium
What do we know?
The product market is moving to a worldwide market
The interpretation of the Halal guidelines is not necessary the same
everywhere
For some people, Halal is associated to a brand and not to a concept
A product formulation can be simple or complex from dozen of
suppliers. How to control all of these suppliers?
Different pork DNA/proteins detection kits exist but their performances
varies widely.
Suspicions of frauds or contaminations of Halal products by pork
tissues are frequently reported
Which is the acceptable cutoff values to be claim free or not free of
pork?
Is it 0.1% or 0.01 % or 0.0001 or 0.00000 ……??
If not free, which is the percentage of
pork in the product?
How much the detection test cost?
Is the product Free or Not Free of Pork?
Can I use the test directly
at the supermarket or only laboratory
is able to do it?
Faced to this situation ( various choice of food, cosmetic or pharmaceutical
products), the consumer is completely powerless.
In the respect of the Halal guidelines, particularly concerning risk of contamination
by pork products, the consumer has 4 central questions
How to be sure of my process and the
respect of Halal/HACCP/ISO/BRC guidelines?
How to guarantee my products?
How to control my suppliers?
Is a problem (contamination, trace) is
discovered on one of mine product,
which corrective measures I will introduce
to solve the problem?
I change the supplier
I change my process
For the manufacturer, the questions are:
How much the detection test cost?
How to determine the cutoff values to decide
what is a fraud, a contamination or a trace?
How to guarantee to our population the highest security
of the products and the respect of Halal precepts?
What standards should be applied in our Halal guidelines
and how to control the respect of these standards?
Are we adopt only a repressive position in a case of
contamination or a constructive position by helping
the manufacturer to adopt corrective solutions?
For the authorities, the questions are:
Which method give us the highest guarantee of
enforcing these standards?
How the informations are diffused between
laboratories, between laboratories and authorities
and finaly, between authorities and the consumers?
How much the detection test cost?
S
S
S
ave = Yes no pork DNA is used as standard reference
minimal risks of contamination (one step test)
functional and easy
Kit is certified Halal by HCQ (NL)
imple = Yes one step ready to use
direct quantification without additionnal manipulations
ure = Yes highest sensibility (0.0001%),
highest specificity (Suidea and Vertebrate)
robustness (different forms of food, pharmaceutical
and cosmetic products)
direct quantification
For the detection of pork DNA, the Assets of the
Progenus TagPro Pig DNA Quantification kit are
To be recommended to or by authorities, a detection kit must to meet
the requirements of the 3 S theory.
Detect protein
Cheaper for single detection
Low Sensitivity (0.1% exogenous protein detected)
Risk of false positives or negatives
Risk of cross-reactivity with proteins of other species
Affected by food thermic treatment
Results not always reliable
The results is obtained in just 15
minutes
Disadvantages of this method:
• Poor Precision
• Low sensitivity (max 0.1 %)
• Low resolution
• Results are not expressed as numbers
• Not always adapted for cooking products
• Risks of false negatives/positives
+ - ? +
Near infrared spectroscopy determine inter-
species differences in the expression of myosin light
chain (MLC) isoforms to identify meat
Disadvantages of this method:
• Only on food sample
• Low sensitivity (max 0.5 – 1 %)
• Results are not expressed as numbers
FP1M
FP4 Contrôle négatif
Cassoulet Contrôle
porcFP1
MFP4 Contrôle
négatif
Cassoulet Contrôle
porc
Chicken cassoulet contamined by pig DNA
Pig DNA
control
Negative
control
Disadvantages of Traditional PCR:
• Poor Precision
• Low resolution
• Non-Automated method
• Results are not expressed as numbers
• Ethidium bromide for staining is not very
quantitative
• Post-PCR processing
• Risk of lab contaminations by ethidium
bromide
Measures the amount of accumulated PCR product
at the end of the PCR cycles.
This method is highly specific with
good sensitivity (0.01 %)
Advantages of Real-Time PCR:
• Increased dynamic range of detection
• No post-PCR processing
• ready to use
• highest sensitivity at the present time (0.00001%)
• highest specificity
• quantification of DNA
This method measures PCR amplification as it occurs.
This method is quantitative, because data is collected
during the exponential growth phase of PCR
The fluorescence is measured during each cycle and the
amount of fluorescence expressed is proportional
to the amount of product.
.
Item ELISA Immuno Chroma
NIR PCR qPCR qPCR Progenus solution
Target Proteins Proteins Proteins DNA DNA DNA
Sensibility 0.1 % 0,1 % 0.5% 0.01 % 0.001 % 0.00001 %
Specificity Not always Not always Pork Pork Pork Suidea
Robustness ± ±
Only food Yes Yes Yes
Expression of the results
Values Signal
Signal
Signal
Ct Value Ct Value
Quantification Standard curve
No No Semi-quantitative
Standard curve
Internal quantification marker in the
same tube
Risk of false negative/positive results
Yes Yes Yes No No No
Rapidity -2 hours 15 minutes 1 hour 4 hours 2h30-3h 2h00
• To produce an One step ready to use kit with minimal
manipulation
• Development of an internal qualibration control (vertebrate)
• No use of pork DNA to construct standard curve
• Quantification of pork DNA in the sample
by comparison with the total vertebrate DNA
in the sample
• Direct detection and quantification of pork
DNA in the sample
Our objectives in the development of a qPCR kit were:
Our strategic process to develop the kit is:
• bioinformatics analyze of the target genome
• specific probes design
• specificity, sensibility, robustness validation
• practical validation
Using our process (bioinformatics analyzes, probe design, specificity, sensibility
and robustness validation, and practical validation) , we develop not only Pork
detection kit but alsoalso other kits
All GMO detection (soon)
Chicken, turkey, ….
Campillobacter spp, E.coli spp, E-Coli H104,….
Avian influenza A/H1N1
…..
30 samples collected in different supermarkets
All samples were labelised Halal certified
qPCR method to detect the Pork DNA
The results were:
16 samples were free of pork
12 samples were contaminated (less than 0.1 %)
2 samples were used from fraud (more than 20%)
Conclusion : without high scientific method, it is difficult to certify a product.
and then, the scientific data are a valuable information for
Halal certification
Item Progenus TagPro detection kit
Provider 1 Provider 2 Provider 3 Provider 4
Sure Highest
Simple One step Ready-to-use
Needs preparation
Needs preparation
Needs preparation
One step Ready-to-use
Save IPC + Vertebrate IPC + EPC IPC + EPC
IPC + EPC
IPC + EPC
Rapidity 2 hours 2h30-3h00 2h30-3h00 2h30-3h00 2h30-3h00
Competitivity < 5 copies < 5 copies 10 copies 10 copies < 5 copies
Sensibility 0.00001 % 0.001 % 0.01 % 0.01 % 0.001 %
Specificity Suidea + Vertebrate Pig Pig + Animal ( ?) Pig + Animal (?) Pig
Quantification Direct & Immediate
Standard curve 16 points
Standard curve 16 points
Standard curve 16 points
Standard curve 16 points
Coverage Food, pharmaceutics,
cosmetc
Food, pharmaceutics,
cosmetc
Food, pharmaceutics,
cosmetc
Food, pharmaceutics,
cosmetc
Food, pharmaceutics,
cosmetc
Training Free / / / /
Support Free
/ / / /
ERP Free / / / /
Price Lower with quantification
Elisa versus qPCR Progenus TagPro
Detection cost
Quantification cost
Number of kits
used in
sanitary
control
>
80 % (2012) 20 % (2012)
20 % (2015) 80 % (2015)
For example: last week, FDA recommends qPCR as the standard method for
quality control in vaccine production.
(5-6 standards +
blanco + sample) x
2 tubes/point = 16
test reactives/
analyse
(1 positive, 1
negative controls +
1 samples) = 3 test
reactives/ analyse
This service issue from the qPCR test, responds to the Halal certification’s needs
in terms of:
Progenus qPCR test applied to Halal certification is not only a scientific result
but also a flexible, complete and high-quality service for the consumer,
manufacturer and authorities.
Security
our qPCR kit is able to detect and to directly quantify
the target with high specificity and sensibility
Position adopted by FDA, for example, clearly indicates that qPCR
is the method that must be recommended for control in routine in
many cases
Progenus qPCR detection test is integrated in a global service
Proof
More and more products are certified Halal. Unfortunately, daily
experiment shows that it is necessary to prove it.
Scientific control must guarantee the product but also to
detect the errors. Our qPCR kit is able to do it
Responsibility
To the consumers, producers and authorities are responsible
of the food quality and security.
our qPCR kit is efficient to control the critical points of industrial
process, logistic and distribution network.
Respect of values
qPCR analyze helps allay consumers' concerns about the correct and not
misleading information on the product label.
« Made in » with quality is important in manufacturing
In front of the trafic products, it is extremely important to be sure that the product is the
real product in the real packaging with the real brand
Our goals: Develoment of a Partneship to input a control certification of the manufacturing and packaging process and its terms of reference
Proof Respect Reponsi- bility
Security
Food of animal origine
Food additives
Preparation, processing, packaging,
transportation, storage
Additional labelling
requirements
ERP
Control, marketing and development
Communication network (laboratories, authorities, …)
CONCLUSIONS
Scientific control by using an efficient method (qPCR Progenus DNA quantification kit)
provides additional important information to certification.
It offers tailored solutions, in keeping with the special characteristics of each industry
certification organism, authorities.
It contributes to a high certification level.
It offers a competitive price for simultaneous detection and quantification of pork DNA.
It provides data that are filtered and transformed into certification, management or
business information, then forwarded to the ERP available for all partners.
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27
Quality control is the real scientific tool of regulation value chains in compliance with the requirements of consumers
Mission of Progenus
28
29
The cycle at which the
amplification plot crosses this
threshold (= cycle threshold or
Ct) is proportional to the initial
amount of target sequence. Baseline
The fluorescence is measured during each cycle
and the amount of fluorescence expressed is
proportional to the amount of product.
We use the software, BioXpress developped in our laboratory
for aligment of available sequences (more than 16 109 informations in our data bank)
for identification of a sequence specific for Suidea (for example, to control of the absence
of cross reaction between our probes and donckey)
for identification of a common sequence of all knowed vertebrate species
to design specific probes for Suidea and vertebrate
Analysis of 16 million of informations
Selection of DNA
fragments what we
want and
rejection of all
false positives
DNA fragments
that could be
assimilated
Mixed meat containing 0.0001% pork (ct = 38,5 )
TagPro Sensibility
Validation by using our own electronic PCR method
of the theorical qPCR conditions (salt, temperature, cycles, …)
Species Suidea probe
Vertebrate probe
Species Suidea probe
Vertebrate probe
Pig + + Fishes - +
Wild boar + + Dairy milk - +
Warthog + + Eggs - +
Cattle - + Wheatmeal - -
Sheep - + Potato - -
Goat - + Tomato - -
Horse - + Aubergine - -
Donkey - + Mushrooms - -
Chicken - + Garlic - -
Duck - + Onions - -
Birds - + Olives - -
Dog - + Artichoke - -
Cat - + Rocket - -
The performance is dependent of the quality of input material (probes,
primers) but also of sample DNA preparation methods.
Dilution Ratio of
pork meat
TagPro Provider 1 Provider 2 Provider 3 Provider 4
1 + + + + +
10 + + + + +
100 + + + + +
1 000 + + + + +
10 000 + + + + +
100 000 + + + + +
1 000 000 + - - - -
10 000 000 + - - - -
The PIG PCR limit of detection is 5 copies of DNA.
Copy number/PCR Eff.
107 106 105 104 103 102
Operator 1
15.00 18.35 22,10 25.26 29.26 31.68
97.45
Operator 2
15.46 18.64 22,33 25.41 29.14 32.46
96.06
Operator 3
15.18 19.00 22,22 25.63 29.02 32.29
96.84
Oper.ator 4
15.47 19.12 22,26 25.71 29.06 32.00
100.50
Robustness 98.86
6 dilutions of a standard Pork DNA
4 different operators
Standard curve versus direct quantification
Mixed meat containing 0.0001% pork (ct = 38,5 ± 1) Mixed meat containing 10 % pork (ct = 22 ± 1)
Positive pig result in coconut oil certified Halal
Vertebrate
DNA IPC
control
PIG DNA
Halal certified product
From a test
to a kit
The kit contains three PCR systems:
-one for the detection of a Suidea specific gene
-one for the detection of a Vertebrate gene
-one for the detection of an internal positive control (IPC)
The three PCR systems are present in
a ready-for-use PCR mastermix allowing
the realization of the
three assays in a single reaction.
Suidea +
Vertebrate +
IPC PCR system
One reaction’s tube
per analyse
Adding 20 µl
of the mastermix
solution
The kit contains three PCR systems labeled with three different dyes in order to allow the
simultaneous quantification of the three targets.
dye
Suidea detection FAM
Vertebrate detection Vic
IPC detection Cy-5
Complex matrice Simple matrice
Automated extraction (limited raw material volume
in the sample)
IPC 5µl
Manual extraction (more raw material volume
in the sample)
+
+ 711
possible combinaisons
to produce the same biscuit
11suppliers/ingredient 7 different ingredients
More complex and
sophisticated is a
product, more
difficult is to
control
the suppliers and
the inputs
CONCLUSION
………… to a complex network
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