Method Verification of the LOD Associated with PCR Approaches for the Detection of Horse Meat
Eloise Busbya and Malcolm Burnsa,b
(a) LGC, Queens Road, Teddington, Middlesex, TW11 0LY, UK
(b) To whom correspondence should be addressed
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In 2013, the Department for Food and Rural Affairs (Defra) and the Food Standards Agency (FSA) commissioned a UK Survey of beef products as part of a co-ordinated response to the EU horse-meat issue. Samples were taken on a formal basis, allowing UK Public Analysts to choose which methods to apply. A range of analytical methods were available for detection of horse DNA, but the respective Limits of Detection (LOD) were often different, not robustly defined, or expressed using different measurement units. The LOD of methods used in the UK Survey needed to be robustly tested and qualified so that results obtained from the samples could be interpreted with confidence.
The aim of the present study was to evaluate the LOD of three selected methods used by Public Analysts as part of the UK horse-meat Survey in terms of uniform w/w (raw horse-meat in a raw beef (meat) background) sample measurements. The three methods evaluated were a PCR-Capillary Electrophoresis approach (PCR-CE as described in Defra project FA0220, LOD reported as approx 1% w/w); PrimerDesign (LOD of approx. <100 mitochondrial copies); and Neogen BioKits (LOD approx 0.1% w/w).
A range of gravimetrically prepared raw horse-meat in raw beef meat (w/w) materials were produced as part of the current study and authenticated for species identity. These materials were used to challenge the three methods in order to estimate the LOD in terms of w/w (meat to meat) based on internationally accepted guidelines and best measurement practice for LOD and PCR methods. Estimates for the LOD were based upon 60-115 replicates of the 0.1% w/w material, depending upon the method evaluated. More than 250 replicates of the 0.1% w/w material were assessed across the three analytical methods, representing five independent DNA extractions.
Results showed that all three methods were capable of reaching an LOD of less than 0.1% w/w raw horse-meat in a raw beef (meat) background if Quality Procedures and Good Laboratory Practice for molecular biology methods were adhered to. This helped afford good comparability of results for these three methods, and in turn contributed to ensuring that the results from the UK Survey of beef products in 2013 were interpreted with confidence.
Horse meat, Limit of Detection (LOD), PCR, real-time PCR, Capillary electrophoresis, Defra project FA0134
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