Development of a novel electrochemical assay for the rapid detection of pathogenic fungi and identification of clinically relevant Candida species

摘要

A number of fungal species, particularly Candida species, are opportunistic pathogens capable of causing disease in humans. These range from relatively mild infections in healthy individuals (e.g. thrush) to life threatening systemic infections and colonisation of major organs in immunocompromised individuals. Existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. Atlas Genetics have developed a novel electrochemical assay for the detection of nucleic acid from target pathogens using PCR followed by hybridisation by labelled probes. The work describes the development of a suite of probes and optimisation of reaction conditions for the rapid detection of fungal pathogens using this assay. A suite of five species-specific probes was developed to detect the five most clinically relevant Candida species as well as a pan-fungal probe capable of detection of DNA from any fungal species. Additionally, since the assay was novel, the work investigated other aspects such as probe and primer design which led to the development of a quick bioinformatics approach for design of species-specific probes which is also described. The results demonstrated that species-specific detection of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei was possible, and that there was no cross reactivity exhibited by any probe with isolates from a test panel of other Candida species. The limit of detection of the assay was shown to be approximately one genome in 1ml of blood or 10 whole cells in 1ml of blood. The use of solid-state electrodes for detection provides an opportunity for miniaturisation of the assay into a robust, easily operated, portable system capable of rapid detection of pathogenic DNA in clinical samples either at point of care or in the microbiological laboratory.