Semi-nested multiplex PCR enhanced method sensitivity of species detection in further-processed meats

Techniques for species assay are potent tools for the supervision of meat adulteration. Matsunaga et al. have established a reliable multiplex PCR method to identify chicken, beef, pork and mutton species in meat products. However, this method was not sensitive enough in the assay of further-processed meats. Here, we attempted 2 strategies, semi-nested multiplex PCR and shortening primers, to enhance the sensitivity of multiplex PCR. As the semi-nested multiplex PCR, first PCR was performed by a pair of common primers, and the product was used as the template of second multiplex PCR. This method lowered the limit of detection (LOD) of multiplex PCR by 3 orders of magnitude, and effectively identified meat species in further-processed foods. The LOD of semi-nested multiplex PCR reached 1 pg of DNA per reaction, which was 10-fold lower than a standardized Real-time PCR method. In contrast, multiplex PCR using truncated primers could hardly meet identical efficiency on different templates, and failed to improve the method sensitivity. The semi-nested multiplex PCR established in this study would be practical in the control of meat adulteration, and could benefit QC of meat manufacture.

► Multiplex PCR (MP) was not sensitive enough to identify further-processed meats. ► Compared to MP, Semi-Nested multiplex PCR (SNMP) greatly enhanced method sensitivity. ► The LOD of SNMP was 10-fold lower than a standard Real-time PCR method. ► Truncating primers failed in improving the sensitivity of MP.