Regular ArticleRapid detection of G6PD mutations by multicolor melting curve analysis

- The clinical and analytical performances of the MeltPro G6PD assay were investigated.
- Comparison studies with biochemical assays and DNA sequencing were performed.
- A novel mutation in the G6PD gene has been found in clinical evaluation.

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was < 0.3 °C. The limit of detection was 1.0 ng/μL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150 min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings.