Defining the performance parameters of a rapid screening tool for myotonic dystrophy type 1 based on triplet-primed PCR and melt curve analysis

<p><b>Background:</b><i>DMPK</i> CTG-repeat expansions that cause myotonic dystrophy type 1 (DM1) can be detected more rapidly, cost-effectively, and simply by combining triplet-primed PCR (TP-PCR) with melting curve analysis (MCA). We undertook a detailed technical validation study to define the optimal operational parameters for performing bidirectional TP-PCR MCA assays.</p> <p><b>Methods:</b> We determined the assays’ analytic specificity and sensitivity, assessed the effect of reaction volumes, DNA diluents, and common contaminants on melt peak temperature, determined the assays’ sensitivity in detecting low-level mosaicism for repeat expansion, and evaluated their performance on two real-time PCR platforms.</p> <p><b>Results:</b> Both assays were highly specific and sensitive, and performed optimally under a broad range of parameters. Bidirectional TP-PCR MCA analysis also reduces the risk of generating false-negative results associated with the rare CCG-interruptions that may be present at either end of expanded alleles.</p> <p><b>Conclusion:</b> The <i>DMPK</i> TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.</p>