Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

The Journal of Molecular Diagnostics : JMD
Grace X Y LimMing Guan

Abstract

Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%-100%) and 99.6% specificity (95% CI, 98.5%-99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates post-PCR ...Continue Reading

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Citations

Oct 22, 2015·Expert Review of Molecular Diagnostics·Flora Tassone
May 7, 2020·Clinical Chemistry and Laboratory Medicine : CCLM·Dong-Sheng LianYan-Yi Wang
Apr 20, 2019·Frontiers in Veterinary Science·Anja JoachimNicola Palmieri
Feb 23, 2021·Intractable & Rare Diseases Research·Nydia Rena Benita SihombingSultana Mh Faradz

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