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Method Validation of the real-time polymerase chain reaction (PCR) approach for the quantitation of horse DNA - FA0146

Food authenticity and food fraud are becoming increasingly prevalent within the European food industry partly due to the pressures faced by producers within today’s challenging financial climate and the international nature of modern food production. The recent EU wide issue involving the detection of the undeclared presence of horse-meat in beef products destined for human consumption has emphasised the need for the development of accurate analytical approaches for the quantitative detection of meat adulteration in a sample.

In response to the horse-meat issue, a real time PCR method was developed at LGC for the quantitation of horse DNA (Defra project FA0135). It was demonstrated that that this assay was capable of detecting and accurately determining the amount of horse DNA present in beef meat products, and the method was validated through objective assessment of performance characteristics using DNA:DNA ad-mixtures and w/w raw horse-meat in raw beef (meat) materials.

In order to enable adoption of the method for use by Public Analysts in a regulatory capacity, including a Knowledge Transfer event and potential Collaborative Trial of the method, the AMWG recommended further validation against the following three criteria:

1) Verify the applicability of the method to different horse samples.
The original method used horse DNA derived from the same horse sample for both the calibrant and the test sample, and it was recommended that the approach be applied to samples from different horse specimens to demonstrate the scope of the method.

2) Evaluate the method’s likelihood that it can detect target species at the 1% level.
The EU have recently published guidance on how to evaluate test samples for determination of horse-meat content above or below a de-minimus labelling threshold of 1%, and it is recommended that the real-time PCR method for quantitation of horse DNA be applied in this context to determine the approach’s capability to accurately quantify samples above and below this threshold.

3) Assess the suitability of the method for use in complex foods.
The real-time PCR method for quantitation of horse DNA will be applied to a limited range of complex and processed food materials, in order to provide evidence of its applicability in quantifying/detecting horse DNA in comparison with other available approaches for detection of horse DNA (e.g. commercial kit).

Qualification of the applicability of the real-time PCR method for quantitation of horse DNA (FA0135) will be assessed through additional method validation addressing the above three criteria. Following this qualification, the AMWG has recommended that the approach be immediately considered for a Knowledge Transfer exercise to disseminate expertise to Public Analysts. This would then position UK Public Analysts to rapidly apply the approach and to participate in associated Collaborative Trials to fully evaluate the reproducibility of the method.
Further limited but focused validation of the real-time PCR method for the quantitation of horse DNA (Defra project FA0135) will benchmark the performance of the method and provide additional evidence of the fitness for purpose of the method in its application to different sources of horse-meat, processed and complex food materials, and its ability to detect and quantify horse-meat around the current de-minimus 1% threshold. This will allow Defra and the FSA to make an objective judgment on the suitability and appropriateness of taking the method forward for potential use and knowledge transfer to UK enforcement laboratories (Public Analysts), challenge exercises with blind samples, and possible collaborative trial of the method at national and international level as an analytical test for the detection and quantitation of horse-meat. This would further allow food companies to make informed decisions on their supply chain based on accurate results which will help to identify the source of the adulterant. Regulators will also be able to confidently enforce labeling laws in cases where this method identifies a non-compliant result. Having a fully validated, quantitative method for the determination of horse DNA in beef based meat products will help regulators to enforce this UK/EU legislation and enable honest traders to robustly defend their food supply chain.
Project Documents
• FRP - Final Report : FA0146 draft final report v03 publication version   (592k)
• OTH - Other : FA0146 SOP publication version   (347k)
Time-Scale and Cost
From: 2013

To: 2014

Cost: £45,053
Contractor / Funded Organisations
LGC Limited
Food Chain              
Food Ingredients