Interlaboratory validation of two multiplex quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk proportions in cheese

Milk products like yogurt, flavoured milk-drinks, curd and cheese may be composed of milk different from cow, namely of ruminant species like sheep and goat. Such products experience an increasing demand in Europe and are recognised as healthy and naturally finished specialities. To verify declared milk compositions in these dairy products, two different quantitative multiplex PCR systems have been evaluated in a comparison test with eleven participating laboratories employing two unknown, traditionally manufactured cheeses with different degrees of ripening to determine milk fractions from cow, ewe and goat. Precision and accuracy was investigated by calibration to dilutions of DNA mixtures and to homologous matrix-adapted reference cheeses, respectively. As expected, independent of the particular method, best inter- and intra-laboratory accuracy has been achieved through the use of homologous reference cheese standards. Furthermore, it has been shown that cheese ripening and the concomitant DNA degradation exert an inverse effect on the method's sensitivity and performance characteristics. Additionally, a broad market survey of different milk products demonstrated its applicability as an efficient analytical tool for food control laboratories to challenge the authenticity of milk and its products from small ruminants.