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|Title:||Development of a macroarray based on 16S-23S rDNA probe hybridization for rapid diagnosis of human pathogenic bacteria|
Kashyap, V K
|Abstract:||The rapid identification of bacteria in biological specimens is important for selection of a suitable antimicrobial therapy. We have designed a rapid (<8 h) diagnostic system that uses universal PCR primers to amplify a variable region of bacterial 16S & 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides, spotted on nylon membrane macroarray slides. Culture dependent assays were used as reference methods in the development and evaluation of this diagnostic platform. Broad range PCR primers were selected from the published literature to amplify 16S & 23S rDNA segments. Species-specific probe sequences were designed based on sequence alignment of eight different bacteria. These species included Helicobacter pylori, Salmonella typhimurium, Shigella dysenteriae, Vibrio choleriae, Neisseria gonorrhea, N. meningitidis, Corynebacterium diphtheriae and Haemophilus influenzae. To verify specificity, five to six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on nylon membrane macroarray glass slides using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were selected based on hybridization results. This procedure was successful in discriminating a range of bacteria in pure cultures. Adding further oligonucleotides to the panel without significantly increasing the cost the accuracy, range, and discriminatory power of the assay can be extended. This method is versatile and makes it possible to detect a large number of bacterial species in a single assay and discriminate different bacterial genera of medical importance. Our results provide a proof of concept for the diagnostic use of macroarray technology based on broad-range ribosomal RNA gene amplification, followed by hybridization and specific detection of bacterial species.|
|Appears in Collections:||IJBT Vol.07(4) [October 2008]|
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