关键词: 肉类掺假, 循环阈值, 碱基错配, 非特异性扩增, 引物特异性

Abstract: Primer specificity is one of the key figures of merit for the identification of meat adulteration by polymerase chain reaction (PCR). In this study, the pattern of occurrence of non-specific amplification in real-time quantitative polymerase chain reaction (RT-qPCR) was evaluated in terms of the ΔCt value between base pair matches and mismatches. Three independent variables including the type, position and number of base pair mismatches were investigated for building a quadratic polynomial to predict the ΔCt value. Results showed that the type, location, and number of base pair mismatches had a significant impact on non-specific RT-qPCR amplification in a very regular pattern. As far as different types of base pair mismatches were concerned, nonspecific amplification between purine and pyrimidine, but not between different purines or between different pyrimidines, occurred easily, with a significant difference being observed between two types of base pair mismatches. For base position, the first base of the 3’ end of the primer had the greatest impact on the ΔCt value; the mismatch position gradually far away from the 3’ end had a decreased effect on the Ct value, making nonspecific amplification occur more easily. The greater the number of base pair mismatches was, the more significant the influence on the ΔCt value was, leading to reduced occurrence of nonspecific amplification. The prediction model for ΔCt was fitted using a stepwise method with correlation coefficient of more than 0.80, which could provide a useful and accurate method to predict the ΔCt value within certain limits.

Key words: meat adulteration, cycle threshold value, base pair mismatch, nonspecific amplification, primer specificity