Development of Common Primer Multiplex PCR (CP-M-PCR) for species-specific meat detection

Abstract

IntroductionA Common Primer Multiplex PCR (CP-M-PCR) was developed to detect three groups of animal species (swine, ruminant and pseudo-ruminant) from meat-based products. This method demonstrated a higher sensitivity and efficiency than the conventional multiplex PCR. ObjectiveTo develop a highly sensitive multiplex PCR method for rapid and accurate identification of meat in meat-based products. MethodsIn this study, a common forward primer was designed at a homologous region of mitochondrial NADH-dehyrogenase subunit 4 (Nad 4) gene sequences of all the animal groups. Adapter reverse primers were designed by adding an adapter sequence at the 5'-end of the specific reverse primers. PCR were performed on DNA extracted from muscle tissue samples using a common forward primer, adapter reverse primers and an adapter primer targeting sequence of Nad 4 gene. A serial of dilution of each reverse primer was used to determine and compare the sensitivity of CP-M-PCR to conventional multiplex PCR system. The detection limit of CP-M-PCR was evaluated with 10-fold serial dilutions of DNA concentration mixture in different ratios of concentration. Results & DiscussionThe use of adapter sequence at the 5'-end of the reverse primers increased the efficiency of the amplification and the application of a single forward primer solved the complexity in multiplex PCR system. Bands of specific amplification can be detected from the PCR assays containing as low as 10–6 μM of adapter reverse primer. This result indicated the sensitivity was tremendously increased as compared to the conventional multiplex PCR (10–3 μM). The limit of detection was as low as 1 ng of DNA. ConclusionCP-M-PCR has greatly improved the sensitivity and efficiency of the PCR system for detecting fraud in meat-based products, resulting in more reliable and accurate results than conventional multiplex PCR system.