Direct lysis-multiplex polymerase chain reaction assay for beef fraud substitution with chicken, pork and duck

Abstract

Since the "Horsemeat Scandal" in Europe has aroused people's attention to adulteration of meat, real-time PCR method was used as gold standard to detect single animal species. However, real-time PCR requires expensive equipment, professional personnel, and fluorescent probes with specific modifications in addition to primers, which limit the practical application of this technology for multiple fraud identification. Therefore, the rapid, economical, multiple detection technology for the authenticity of animal-derived ingredients is in urgent need. In this study, a novel direct lysis-multiplex polymerase chain reaction (DL-mPCR) method is reported to simultaneously identify pork, chicken and duck adulterated in beef products within 90 min for the first time. A novel direct lysis (DL) method is employed to obtain sample DNA in a more convenient way without a high-speed centrifuge. The sample preparation step is greatly simplified compared to commercial kit method and can be completed within 15 min. Four pairs of high-specificity primers for the mitochondrial D-loop region of cattle, pig, chicken and duck were designed. The highly specific and sensitivity mPCR assay with optimal primer ratio was constructed, which could detect pork, chicken and duck down to 0.1% (w/w) in meat mixtures treated with freezing, heating and autoclaving, respectively. It was found that 27.8% of 79 commercial beef products collected in local markets contained pork or chicken to varying degrees. The accuracy of the results had been verified by standard real-time PCR. This novel approach (DL-mPCR) proved to be very reliable, time-saving and practical, especially suitable for low-resource front-line detection of meat authentication.