Abstract:The research aimed to establish a Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergen β-lactoglobulin in food. Specific primers and Taqman probe were first designed based on the gene sequence of β-lactoglobulin for real-time PCR. The purified DNA product was linked with the pMDl9-T vector to construct recombined plasmids as a standard PCR template. Plasmids were then identified with colony PCR and subjected to sequencing. Real-time fluorescence PCR assay was performed with plasmids and the standard curve was constructed with DNA copies and Ct. Sensitivity, specificity, reproducibility and application of the real-time method were also evaluated. Results showed that the β-lactoglobulin DNA fragment was successfully cloned and the standard curve had a good linear relationship ranging from 3.18×103 to 3.18×107 copies. The detection sensitivity reached up to 318 copies/μL. Furthermore, specificity and stability of the method were good. Ten foods were detected with the β-Lg DNA residue and the results were consistent well with their allergen labels. Therefore, the method may become a complementary tool for milk allergen detection and it is applicable for other food allergens.
