Differential detection of sheep and goat meat using duplex real-time PCR and high-resolution melt analysis

Abstract

Authenticity in meat and meat products is of paramount importance in preventing adulteration and ensuring food safety. Since morphologically indistinct samples like sheep and goat meat requires accurate and sensitive differentiation, we developed and validated a duplex real-time polymerase chain reaction (qPCR) assay with high-resolution melt analysis (HRMA) for simultaneous identification of sheep and goat meat. Species-specific amplicons with melting temperature of 78 °C for goat and 80 °C for sheep (with associated standard errors) was identified. The assay was optimized for basic PCR parameters and the HRMA detection was standardized based on melt curve shape, color and inflexion points. The absolute limit of detection (LODabs) and limit of quantification (LOQ) of the assay were found to be 0.39 ng and 0.78 ng of target DNA respectively. A sensitivity of 0.1% was obtained for both sheep and goat meat. The relative limit of detection (LODrel) was also found to be 5%. The linearity and efficiency were evaluated in both simplex and duplex assays. The assay applicability was evaluated using proficiency test samples and commercially available raw and processed meat samples. The obtained results showed that this closed-tube qPCR-HRMA method can be a rapid and sensitive detection technique for meat speciation.