Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA

Background Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation. Methods Recombinant wild-type and mutant plasmids of c.3321delA mutation were constructed, heterozygous mutant plasmids were prepared by mixing the mutant plasmids and wild-type plasmids at 1:1 ratio. High-resolution melting analysis (HRMA) coupled with an unlabeled DNA probe was employed to identify the shift in melting temperature of the probe–template complex, which reflects the presence of c.3321delA mutation. Results Unlabeled probe based HRMA was able to distinguish all three genotypes (wild-type, heterozygote, and mutant) of c.3321delA mutation. Then, we applied this method to genotype 1,317 clinical samples. Genotyping results obtained from unlabeled probe HRMA were 100% concordant with the results from direct sequencing. Conclusion We developed a fast and high-throughput method to detect the c.3321delA mutation.