Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument.

Scientific Reports
Lingxia JiangPaul Hung

Abstract

Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

Associated Clinical Trials

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Datasets Mentioned

BETA
1

Methods Mentioned

BETA
SMA
genotyping
PCR
electrophoresis
of

Clinical Trials Mentioned

NCT01754441
NCT02532244

Software Mentioned

BLAST
Primer
PrimerQuest Tool
Absolute
Combinati Analysis
Combinati
Absolute Q Analysis
QuantStudio 3D dPCR System
Absolute Q Control
- BLAST

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