Dataset Information

Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

ABSTRACT: Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

SUBMITTER: Bhattacharyya RP

PROVIDER: S-EPMC6418090 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

Bhattacharyya Roby P RP Walker Mark M Boykin Rich R Son Sophie S SS Liu Jamin J Hachey Austin C AC Ma Peijun P Wu Lidan L Choi Kyungyong K Cummins Kaelyn C KC Benson Maura M Skerry Jennifer J Ryu Hyunryul H Wong Sharon Y SY Goldberg Marcia B MB Han Jongyoon J Pierce Virginia M VM Cosimi Lisa A LA Shoresh Noam N Livny Jonathan J Beechem Joseph J Hung Deborah T DT

Scientific reports 20190314 1

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy a ...[more]

PMID: 30872641

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Project description:BackgroundTraditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control.MethodsWe aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.ResultsThe established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.ConclusionsOverall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

Dataset Information

Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

Publications

Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

Similar Datasets

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