Development of a Simple DNA Extraction Method and Candida Pan Loop-Mediated Isothermal Amplification Assay for Diagnosis of Candidemia
To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary.
In the study, it was developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min - the filtration process was performed using a SEPARA® tube (GVS, Bologna, Italy). The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method.
These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.
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Artigo-Vials-SEPARA-e-Tracketched---DNA.pdf