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

BACKGROUND

DMPK 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.

METHODS

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.

RESULTS

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.

CONCLUSION

The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.