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 ……??
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 Is the product Free or Not Free of Pork? If not free, which is the percentage of pork in the product? Can I use the test directly at the supermarket or only laboratory is able to do it? How much the detection test cost?
For the manufacturer, the questions are: How to control my suppliers? How to be sure of my process and the respect of Halal/HACCP/ISO/BRC guidelines? How to guarantee my products? 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 How much the detection test cost?
For the authorities, the questions are: What standards should be applied in our Halal guidelines and how to control the respect of these standards? Which method give us the highest guarantee of enforcing these standards? 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? Are we adopt only a repressive position in a case of contamination or a constructive position by helping the manufacturer to adopt corrective solutions? How the informations are diffused between laboratories, between laboratories and authorities and finaly, between authorities and the consumers? How much the detection test cost?
To be recommended to or by authorities, a detection kit must to meet the requirements of the 3 S theory. For the detection of pork DNA, the Assets of the Progenus TagPro Pig DNA Quantification kit are S ure = Yes highest sensibility (0.0001%), highest specificity (Suidea and Vertebrate) robustness (different forms of food, pharmaceutical and cosmetic products) direct quantification 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) S imple = Yes one step ready to use direct quantification without additionnal manipulations
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
Measures the amount of accumulated PCR product at the end of the PCR cycles. This method is highly specific with good sensitivity (0.01 %) Disadvantages of Traditional PCR: • Poor Precision Chicken cassoulet contamined by pig DNA • Low resolution • Non-Automated method • Results are not expressed as numbers • Ethidium bromide for staining is not very quantitative M M • Post-PCR processing Cassoulet Cassoulet Contrôle Contrôle Contrôle Contrôle FP4 FP4 FP1 FP1 Pig DNA Negative • porc porc négatif négatif control control Risk of lab contaminations by ethidium bromide
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. . 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
Item ELISA Immuno NIR PCR qPCR qPCR Chroma 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 Values Signal Signal Signal Ct Value Ct Value results Quantification Standard No No Semi- Standard Internal curve quantitative curve quantification marker in the same tube Risk of false Yes Yes Yes No No No negative/positive results Rapidity -2 hours 15 minutes 1 hour 4 hours 2h30-3h 2h00
Our objectives in the development of a qPCR kit were: • 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 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 Provider 1 Provider 2 Provider 3 Provider 4 detection kit Sure Highest Simple One step Needs Needs Needs One step Ready-to-use preparation preparation preparation 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 & Standard curve Standard curve Standard curve Standard curve Immediate 16 points 16 points 16 points 16 points Coverage Food, Food, Food, Food, Food, pharmaceutics, pharmaceutics, pharmaceutics, pharmaceutics, pharmaceutics, cosmetc cosmetc cosmetc cosmetc cosmetc Training Free / / / / Support Free / / / / ERP Free / / / / Price Lower with quantification
Elisa v ersus qPCR Progenus TagPro Detection cost << (5-6 standards + (1 positive, 1 blanco + sample) x negative controls + Quantification cost >> 2 tubes/point = 16 1 samples) = 3 test test reactives/ reactives/ analyse analyse Number of kits 80 % (2012) 20 % (2012) used in sanitary 20 % (2015) 80 % (2015) control For example: last week, FDA recommends qPCR as the standard method for quality control in vaccine production.
Progenus qPCR detection test is integrated in a global service 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. This service issue from the qPCR test, responds to the Halal certification’s needs in terms of: 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
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