A Practical Approach to Evaluate the Equivalence of Real-Time Pcr Reagents Used As Substitute in Validated Methods

It is common practice to perform the validation procedure of real-time PCR based methods using commercially available ready-to-use PCR reagents, so called master mixes. In general there is no detailed knowledge about the individual components of these mixes. If changing to a different product-no matter whether a novel mix from the same or a mix from a different manufacturer-this may consequently result in considerable efforts to validate the method again according to the ISO 17025 standard. To minimize the amount of work in such cases a simple procedure to test the equivalence of different master mixes in accordance to the ISO requirements is presented. The procedure allows analyzing the equivalence within a single real-time PCR run in a very simple manner. It is universally applicable and can be applied for any real-time PCR reagent or master mix. The practicability of the procedure is demonstrated presenting experimental data for two different master mixes from test runs applying the event-specific real-time PCR method for the detection of maize MON863 [EURL GMFF http://gmo-crl.jrc.ec.europa.eu/summaries/MON863-WEBProtocol-Validation.pdf (2005)].