2, Hedmark University College, Hamar, Norway
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We have developed multiplex quencher extension (multiplex-QEXT), which is a novel method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The multiplex-QEXT detection is done in real-time by direct measurements of fluorescence changes in a closed tube system. The principle of multiplex-QEXT is that 5′ reporter-labeled probes are 3′ single-base-extended with TAMRA™-labeled dideoxy cytosine if the respective SNP alleles are present. TAMRA can serve as either a fluorescence energy acceptor or donor, depending on the 5′ fluorescent reporter. The extension results in increased reporter fluorescence by fluorescence resonance energy transfer (FRET) if TAMRA serves as an energy donor, while if TAMRA is an acceptor, then the reporter fluorescence will be quenched. The multiplex-QEXT principle is shown schematically in (Figure 1).
Figure 1.
There is a continuous development and improvement of SNP detection technologies (1,2,3,4). Three general strategies have been used to increase efficiency and throughput. Increased throughput has been obtained by pooling the samples and analyzing SNP frequencies in the sample pools (1,5,6). Another strategy has been to develop real-time approaches that require very little sample handling (1,5,7,8,9,10). Finally, multiplex detection of several SNP alleles simultaneously from the same sample is commonly done to increase throughput (11,12,13). To our knowledge, no assay has yet exploited the combined benefit of the three strategies mentioned above (14). The aim of our work was to develop an assay that enables the integration of all the above-mentioned approaches.
Typing of Listeria monocytogenes was used as a model to demonstrate the application of multiplex-QEXT. This pathogenic bacterium represents a major problem for modern food production (15). L. monocytogenes is often persistent in food and food production environments, but only a few types are pathogenic to humans (16). There is, however, a very serious outcome of listeriosis, with a mortality rate above 30%. Determining the reservoirs and understanding food contamination are very important in combating this bacterium (15).
Multi-locus sequence typing (MLST) is emerging as an alternative technique for bacteria typing (17). MLST is based on the DNA sequencing of several different loci in each bacterium analyzed. The identified SNP alleles are then used for the bacterial typing. DNA sequencing, however, severely limits the throughput of MLST. Furthermore, most of the DNA sequence information is redundant. Recently, typing techniques that only target the informative SNP alleles have been developed (1,10). What is lacking, however, are techniques that enable high-throughput analyses of several SNP loci simultaneously.
Here, we demonstrate the high-throughput application of multiplex-QEXT by the detection of four different SNP loci in 252 L. monocytogenes strains. We present data that show the discriminatory power, reproduc-ibility, and quantitative properties of multiplex-QEXT.
Materials and Methods Bacterial Strains and QEXT TemplateWe analyzed 252 L. monocytogenes strains (listed in Supplmentary Table S1, available online at www.BioTechniques.com). The strains were grown overnight in trypton soya broth (TSB) at 30°C, and DNA was purified as previously described (10). All positions of primers, probes, and products are given relative to an inlA reference sequence (GeneBank® accession no. AF497167).
