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Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

Edwards, T, Sasaki, S, Williams, C, Hobbs, G, Feasey, NA, Evans, K and Adams, ER (2018) Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay. Scientific Reports, 8. ISSN 2045-2322

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Abstract

The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively.

Item Type: Article
Uncontrolled Keywords: Science & Technology; Multidisciplinary Sciences; Science & Technology - Other Topics; REAL-TIME PCR; BLOOD CULTURE IDENTIFICATION; SPECTRUM-BETA-LACTAMASES; ANTIMICROBIAL RESISTANCE; PSEUDOMONAS-AERUGINOSA; KLEBSIELLA-PNEUMONIAE; RAPID IDENTIFICATION; RIBOSOMAL-RNA; BACTERIA; GENES
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Nature Publishing Group
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Date Deposited: 16 Feb 2018 12:30
Last Modified: 16 Feb 2018 12:30
DOI or Identification number: 10.1038/s41598-017-18915-5
URI: http://researchonline.ljmu.ac.uk/id/eprint/7964

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