Detección molecular de la variante FQ188R (c.563A>G), gen CFTR cromosoma 7 región 7q31; mediante PCR en tiempo real con tecnología TaqMan™ y verificación con Luna Universal Probe qPCR Master Mix v1

Gene: CFTRChromosomal location: 7q31.2Variant: F508del (ΔF508; p.Phe508del)Nucleotide change: NM_000492.4.1521_1523delCTTdbSNP: rs113993960TaqMan Assay ID: C_151693869_10Application: Research and methodological standardization. Abstract This protocol describes the molecular detection and allelic discrimination of the F508del variant (c.1521_1523delCTT; rs113993960; p.Phe508del) in the CFTR gene using real-time PCR with TaqMan SNP Genotyping Assay technology. F508del is a three-nucleotide in-frame deletion resulting in the loss of phenylalanine at position 508 of the CFTR protein and represents the most common pathogenic variant associated with cystic fibrosis. The procedure includes the selection and in silico characterization of the Applied Biosystems TaqMan SNP Genotyping Assay C_151693869_10, genomic DNA extraction and quality assessment, characterization of genotype controls, real-time PCR amplification using TaqMan Genotyping Master Mix, and allelic discrimination through FAM and VIC fluorescence signals. Amplification with TaqMan Genotyping Master Mix is performed at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Genotype assignment is based on amplification performance, fluorescence intensity, reference controls, and allele discrimination plots. An additional methodological verification is proposed using Luna Universal Probe qPCR Master Mix under the manufacturer's recommended amplification conditions. This complementary experiment aims to evaluate assay compatibility and qualitative genotype concordance between two independent master mix formulations. The protocol provides a standardized experimental framework for identifying homozygous reference, heterozygous, and homozygous F508del genotypes, while considering the availability of genotyped controls, technical variability, and limitations of targeted mutation detection.