title: Bead array direct rRNA capture assay (rCapA) for amplification free speciation of Mycobacterium cultures.
abstract: Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay.
authors.affiliation: KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: de Ronde
authors.forename: Paula
authors.initials: P
authors.lastname: González Alonso
authors.forename: Dick
authors.initials: D
authors.lastname: van Soolingen
authors.forename: Paul R
authors.initials: PR
authors.lastname: Klatser
authors.forename: Richard M
authors.initials: RM
authors.lastname: Anthony
mesh_terms: D001331:Automation; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D001671:Biochemistry / Q000379:methods; D005798:Genes, Bacterial; D017930:Genes, Reporter; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D012335:RNA, Ribosomal / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S