We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Common spreadsheet software was used for automated genotype assignment. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). Emerg Infect dis 4(3), 382–389.Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. (1998) Detection and identification of previously unrecognized microbial pathogens. (2004) Identification of regions in multiple sequence alignments thermodynamically suitable for targeting by consensus oligonucleotides: application to HIV genome. (1999) Multiplex detection of single-nucleotide variations using molecular beacons. (1999) Multiplex detection of four pathogenic retroviruses using molecular beacons. R., Marras, S.A., Tyagi, S., Dube, S., Poiesz, B.J., et al. (2004) Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. A., Templeton, K., Schinkel, J., Brienen E. (1991) Detection of specific polymerase chain reaction product by utilizing 5 ′-3 ′ exonuclease activity of Thermus aquaticus DNA polymerase. (1998) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. (2005) Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR. Guilbaud, M., Coppet, P., Bourion, F., Rachman, C. (2005) Diagnostic system for rapid and sensitive differential detection of pathogens. Key Wordsīriese, P., Palacios, G., Kokoris, M., Jabado, O., Liu, Z., Renwick, N., Kapoor, V., Casas, I., Pozo, F., Limberger, R. allele AllTests Alpha Biosciences Alphabioregen Amoytop Biotech Anogen AnyGenes Ape Apexbio Arbor Assays arista Arthus Biosystems Articular Engineering Artron AS ONE INTERNATIONAL. Here, we describe a program AlleleID that designs qPCR and microarray assays to identify and detect pathogens. Manual design of these primer and probes is both tedious and results in lower quality of results because of the inability to simultaneously handle multiple criteria for design. Both of these techniques have a high accuracy when used with specific primers and probes. Although numerous chemistries are available for the molecular level identification of pathogens, the most common are qPCR and DNA microarrays. Sequence-based molecular methods such as real-time PCR, microarrays, and band biosensors provide high sensitivity, rapid diagnostics, and higher specificity allowing differentiation between related strains. Nucleic acid-based methods for the detection of microorganisms are rapid, sensitive and are generally successful even when the culturing of microorganisms fails. Conventional methods have longer turnaround time and, in most cases, lower sensitivity. Efficient clinical diagnosis of pathogens is important for the management of infectious diseases.
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