The assay uses amplification of the fragile X mental retardation 1 (FMR1) trinucleotide repeat region, followed by a hybridization assay to quantify the number of repeats in the amplicons.

Scientists working in Molecular Diagnostics at PerkinElmer Health Sciences (Waltham, MA, USA) have developed the first repeat-counting assay that uses fluorescent signals rather than electrophoresis or mass spectrometry as the signaling mechanism. They also developed a simple microfluidic electrophoresis reflex test that uses the same amplicons and reduces the need for Southern blots to differentiate homozygous female normal samples from full mutations.

The assay was tested on DNA from 1,008 dried blood spot samples from pregnant women in their first trimester. Reflex testing was performed on the 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA), a compact microfluidic capillary electrophoresis platform, using the Agilent Technologies DNA 7500 kit. Before electrophoresis, the polymerase chain reaction (PCR) products were first purified using the PureLink PCR kit (Invitrogen; Grand Island, NY, USA).

The hybridization assay identified 51 of those as potentially expanded alleles of equal to or greater than 45 repeats or as intermediate or higher in FMR1 repeat classification. Of these screen-positive samples, eight were confirmed by microfluidic electrophoresis as premutations consisting of equal to or greater than 55 repeats. Results for the PCR and hybridization assay are obtained in less time than an eight-hour shift, and the confirmation electrophoresis of screen-positive results takes approximately an additional two hours. All manipulations for the PCR and hybridization assay are performed in microplates and can be performed with a multichannel pipette for high throughput, with the prospect for straightforward automation.

The Luminex 200 (Austin, TX, USA) suspension array system reads the fluorescence results of the hybridization assay unattended. The ratios of fluorescent signals indicative of the repeat length are simple to calculate and embody into software, and no visual interpretations of electropherograms are needed for the approximately 95% of samples that will assay as screen assay negative in most screening settings. The authors concluded that their preliminary results suggest that the assay is a reasonable candidate for screening maternal samples for premutation yes-no status. With appropriate clinical validation, this assay will enable high-throughput, low-cost, low-investment screening. The study was published in the November 2011 issue of the Journal of Molecular Diagnostics.

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