High resolution real-time PCR melting curve assay for identification of top five Penaeidae shrimp species

Abstract In the United States shrimps is the most popular seafood. Penaeidae shrimp species are known to share morphology characteristic and identification based on morphological appearance becomes more challenging when shrimp is processed into ready-to-eat products. Misrepresentation of shrimp is common in commercially available shrimp. The objective of the study was to develop a high-resolution melting (HRM) real time PCR assay targeting the 16S rRNA gene fragment for specific identification of top five penaeid species, Litopenaeus vannamei, Penaeus duorarum, Penaeus monodon, Litopenaeus setiferus and Pleoticus muelleri. The applicability of assay was evaluated using two DNA extraction kits and two real-time PCR master mixes. The HRM assay was evaluated using 43 shrimp samples and results were validated by sequencing shrimp 16S rRNA gene fragment. Assay standardized in this study formed distinct melt curve profile for each species in the normalized and differential melt curve plots. The assay using Apex qPCR 2 × GREEN master mix showed 100% sensitivity and specificity. Further, species identification results obtained by HRM assay was in complete agreement with identification achieved by 16S rRNA gene sequencing. The HRM assay developed in this study can be used as rapid, low-cost, and reliable method for the identification of abovementioned shrimp species.