substances Renaville R. Progenus sa Gembloux, Belgium What do we - - PowerPoint PPT Presentation

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

• f 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

+

• ?

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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

FP1 M FP4

Contrôle négatif Cassoulet Contrôle porc

FP1 M FP4

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

• ur 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

• f the food quality and security.
• ur 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

• f the manufacturing

and packaging process and its terms of reference

Proof Respect Reponsi

• bility

Security

Food of animal

• rigine

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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Quality control is the real scientific tool of regulation value chains in compliance with the requirements of consumers Mission of Progenus

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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

• f 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

• f 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

• f 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