title: Fast and highly specific DNA-based multiplex detection on a solid support.
abstract: Highly specific and fast multiplex detection methods are essential to conduct reasonable DNA-based diagnostics and are especially important to characterise infectious diseases. More than 1000 genetic targets such as antibiotic resistance genes, virulence factors and phylogenetic markers have to be identified as fast as possible to facilitate the correct treatment of a patient. In the present work, we developed a novel ligation-based DNA probe concept that was combined with the microarray technology and used it for the detection of bacterial pathogens. The novel linear chain (LNC) probes identified all tested species correctly within 1 h based on their 16S rRNA gene in a 25-multiplex reaction. Genomic DNA was used directly as template in the ligation reaction identifying as little as 10(7) cells without any pre-amplification. The high specificity was further demonstrated characterising a single nucleotide polymorphism leading to no false positive fluorescence signals of the untargeted single nucleotide polymorphism (SNP) variants. In comparison to conventional microarray probes, the sensitivity of the novel LNC3 probes was higher by a factor of 10 or more. In summary, we present a fast, simple, highly specific and sensitive multiplex detection method adaptable for a wide range of applications.
authors.affiliation: Molecular Diagnostics, AIT Austrian Institute of Technology, Muthgasse 11, 1190, Vienna, Austria, ivan.barisic@ait.ac.at.
authors.forename: Ivan
authors.initials: I
authors.lastname: Barišić
authors.forename: Verena
authors.initials: V
authors.lastname: Kamleithner
authors.forename: Silvia
authors.initials: S
authors.lastname: Schönthaler
authors.forename: Herbert
authors.initials: H
authors.lastname: Wiesinger-Mayr
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D001424:Bacterial Infections / Q000175:diagnosis; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D011088:DNA Ligases; D000072481:DNA Ligase ATP