Lactase persistence genotyping on whole blood by loop-mediated isothermal amplification and melting curve analysis

Abstract

BackgroundThe lactase persistence phenotype is controlled by a regulatory enhancer region upstream of the Lactase (LCT) gene. In northern Europe, specifically the −13910C > T variant has been associated with lactase persistence whereas other persistence variants, e.g. −13907C > G and −13915 T > G, have been identified in Africa and the Middle East. The aim of the present study was to compare a previously developed high resolution melting assay (HRM) with a novel method based on loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) with both whole blood and DNA as input material. MethodsTo evaluate the LAMP-MC method, we used 100 whole blood samples and 93 DNA samples in a two tiered study. First, we studied the ability of the LAMP-MC method to produce specific melting curves for several variants of the LCT enhancer region. Next, we performed a blinded comparison between the LAMP-MC method and our existing HRM method with clinical samples of unknown genotype. ResultsThe LAMP-MC method produced specific melting curves for the variants at position −13909, −13910, −13913 whereas the −13907C > G and −13915 T > G variants produced indistinguishable melting profiles. ConclusionThe LAMP-MC assay is a simple method for lactase persistence genotyping and compares well with our existing HRM method.