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



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.



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.



The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.
a Khoo Teck Puat - National University Children's Medical Institute , National University Health System , Singapore , Singapore.
Mulias
M
Lian
b Department of Pediatric Medicine , KK Women's and Children's Hospital , Singapore , Singapore.|c Pediatrics Academic Clinical Program , Duke-NUS Graduate Medical School , Singapore , Singapore.
Hai-Yang
HY
Law
d Department of Biochemistry, Yong Loo Lin School of Medicine , National University of Singapore , Singapore , Singapore.|e Cancer and Stem Cell Biology Program , Duke-NUS Graduate Medical School , Singapore , Singapore.|f Division of Medical Sciences , National Cancer Center , Singapore , Singapore.
Caroline G
CG
Lee
a Khoo Teck Puat - National University Children's Medical Institute , National University Health System , Singapore , Singapore.|g Department of Pediatrics, Yong Loo Lin School of Medicine , National University of Singapore , Singapore , Singapore.|h Department of Laboratory Medicine , National University Hospital, National University Health System , Singapore , Singapore.
Samuel S
SS
Chong
D000483:Alleles; D002460:Cell Line; D006801:Humans; D009030:Mosaicism; D009223:Myotonic Dystrophy / Q000175:diagnosis* / Q000235:genetics*; D065636:Myotonin-Protein Kinase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019680:Trinucleotide Repeat Expansion
D065636:Myotonin-Protein Kinase
DMPK; MCA; Myotonic dystrophy type 1; TP-PCR; screening