12 Reverse hybridization enables fast and reliable detection of 25 common CF mutations.

Objective Nationwide CF newborn screening (CFNBS) will be established in Germany in 2015. IRT is a reliable marker for the 1 st tier of CFNBS. Although sweat testing represents the gold standard for CF diagnostics, it is of limited reliability in (preterm) neonates. A fast and simple assay to screen for common CF mutations using newborn screening cards would be beneficial. Methods Analysis of 25 common CFTR mutations was performed using the CFcheck EU-25 kit (Astra Biotech) on 66 samples with known CFTR genotype. Fluorescent probes were generated in two consecutive multiplex PCR reactions. Pooled PCR products were hybridized to a slide containing targets corresponding to wild type and mutant CFTR sequences. Signal intensities were measured with a laser scanner. Results The mutation detection rate was 100% (66/66). False positive signals occurred in 5 patients and led to 7.6% repeat analyses. The specificity was 99.8% according to the high number of screened mutations on each sample (25 on 2 alleles/sample). Conclusion The reliable and fast detection of 25 CF mutations using newborn screening cards following positive CFNBS results may complement present CFNBS programs. Early knowledge of genotype, reduced number of recalls and sweat tests may outweigh unwanted heterozygous carrier detection. [ABSTRACT FROM AUTHOR]