The PCR-CE/FA0220 method, the Neogen BioKit and the PrimerDesign method were assessed on comparability of results.
Providing that laboratories apply Quality Procedures and Good Laboratory Practice for DNA extraction and application of molecular biology methods, it is reasonable that the LOD should also be readily achievable, said the analysis.
Establishment of a robust LOD for the methods would allow the results from the Defra/FSA survey on detection of equine DNA in food samples to be interpreted with confidence.
LOD can be the lowest concentration of target analyte that can be detected 95% of the time.
The Department for Food and Rural Affairs (Defra) and the Food Standards Agency (FSA) commissioned a UK Survey of beef products as part of the EU horse meat issue.
Choice of methods
Samples were taken on a formal basis, allowing public analysts to choose which methods to apply.
A range of methods were available for detection of horse DNA, but the respective 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 from the samples could be interpreted with confidence.
Different gravimetrically prepared raw horse-meat in raw beef meat (w/w) materials were produced and authenticated for species identity.
These materials were used to challenge the methods to estimate the LOD in terms of w/w (meat to meat) based on international guidelines and best measurement practice for LOD and PCR methods.
To facilitate the establishment of the LOD, gravimetrically weighted raw horse meat in raw beef (meat) samples were prepared at the 100%, 5%, 1%, 0.5% and 0.1% w/w levels.
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.
Three methods evaluated
Singleplex PCR was carried out for the PCR-CE/FA0220 qualitative approach with 50ng DNA in a volume of 25μl using a Multiplex Master Mix (Qiagen) and a PE9700-9 thermal cycler.
Thermal cycling conditions were used as recommended by Qiagen, and included 45 cycles of denaturation/annealing/elongation. In terms of the LOD, the original method had this as 1% w/w.
Neogen BioKits (HE mastermix pod) method was carried out as specified by the manufacturer5, using the mastermix provided and AmpliTaq Gold DNA Polymerase (Applied Biosystems).
The master mix contains two assays for duplex amplification of a horse-specific DNA target and an unspecified mammalian housekeeping gene target. 50-100ng DNA was added to a total reaction volume of 25μl, and the recommended cycling conditions were followed using a PE9700-9 thermal cycler.
Neogen BioKits protocol states that the LOD of the method is less than 0.1% w/w.
The PrimerDesign real-time PCR approach was conducted using TaqMan Universal PCR Mastermix (Life Technologies) on the ABI Prism 7900HT Real Time PCR system. The primer and probe mix supplied with the PrimerDesign kit was reconstituted in nuclease-free water, prior to the addition of 1μL mix per reaction.
Replicate 20μl reactions were prepared, containing 25ng of genomic DNA extracted from 0.1% w/w horse meat in beef.
These were run alongside the Equus caballus positive control DNA provided with the kit, encompassing the range of 102 to 106 copies mitochondrial DNA per reaction as an approximate guide for DNA amount.
Thermal cycling conditions were: 50°C for 2 minutes, 95°C for 10 minutes, followed by 50 cycles of: 95°C for 15 seconds, 60°C for 15 seconds. The LOD was stated as 100 copies of mitochondrial DNA.
The in-house DNA extraction method was based on cell lysis with SDS, binding of DNA to positively charged beads, and subsequent washing, ethanol precipitation and elution stages.
It was chosen so as to emulate DNA representative of different yield and quality metrics, as the approach used by laboratories in the original UK survey of beef-products was not prescribed.
“Method Verification of the LOD Associated with PCR Approaches for the Detection of Horse Meat”
Authors: Eloise Busby and Malcolm Burns