Multiplex real-time single nucleotide polymorphism detection and quantification by quencher extension

Summary

Multiplex quencher extension (multiplex-QEXT) is a novel closed tube single-step method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The principle of multiplex-QEXT is that 5' reporter-labeled probes are 3' single-base-extended with TAMRAT (TM) dideoxy nucleotides if the respective SNP alleles are present. TAMRA can serve as either an energy acceptor (quencher-based detection) or donor [fluorescence resonance energy transfer (FRET)-based detection] for a wide range of different reporter fluorochromes. The extension can therefore be recorded by the respective reporter fluorescence change. We evaluated multiplex-QEXT analyzing four different SNP loci in the Listeria monocytogenes inlA gene. Probes labeled with the reporters 6-FAM (TM), TET (TM),, VICT (TM), and Alexa Fluor((R)) 594 were used. Responses for the fluorochromes 6-FAM, TET. and VIC were detected by quenching (decreased fluorescence), while the response for Alexa Fluor 594 was detected by FRET (increased fluorescence). tire evaluated the SNP-allele pattern in 252 different L. monocytogeties strains. Multiplex-QEXT gave a good resolution, detecting seven major and five minorgroups of L. monocytogenes. Comparison with serotyping showed that multiplex-QEXT gave better resolution. We also evaluated the quantitative aspects of multiplex-QEXT Quantitative information was obtained for all the fluorochrome/probe combinations in the sample pools. The detection limits for 6-FAM, TET and Alexa Fluor 594 were the presence of the 10% target SNP alleles (P < 0.05), while the detection limit for VIC was the presence of the 5% target SNP alleles (P < 0.05). Currently overlap in the fluorescence emission spectra is the limiting factor for the multiplexing potential of QEXT With the emergence of new fluorochromes with narrow emission spectra, we foresee great potential for increasing the multiplex level in the future.