ABSTRACT
Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.