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Modified and Improved DNA Extraction Method for Molecular Detection of Candida Species from Positive Blood Culture Bottles
EP31084
Poster Title: Modified and Improved DNA Extraction Method for Molecular Detection of Candida Species from Positive Blood Culture Bottles
Submitted on 11 Dec 2019
Author(s): Yahaya Hassan and Leslie Thian Lung Than
Affiliations: 1Department of Medical Laboratory Science, Faculty of Allied Health Sciences, Bayero University Kano, Nigeria. 2Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Selangor, Malaysia.
This poster was presented at ASEAN Emerging Researchers Conference 2019, Sunway University Malaysia.
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Poster Information
Abstract: Background and Objective: Yeast DNA extraction from positive blood culture bottles is often difficult and challenging due to the presence of many inhibitory factors. Among them include abundant interfering human DNA, blood preservatives used in blood culture bottle, blood cells components such as haem and the complex and sturdy nature of the yeast cell wall itself. These factors frequently result in getting negative results in laboratory molecular detection. To validate a modified and improved ‘‘in-house’’ method for extracting yeast DNA directly from blood culture bottle specimens which is rapid, simple, safe and economical for identification of Candida species. Methods: Six positive blood culture bottle specimens were used for DNA extraction using modified conventional method based on boiling method (thermal lysis) (95°C for 30 min), precipitation of the DNA using sodium acetate and absolute ethanol, followed by DNA purification using a general commercial kit, all within 1 h 20 min. The method does not require the use of lysis buffer, proteinase K or lyticase enzymes, or harmful phenol-chloroform chemicals, or the use of expensive commercial DNA extraction kit for extraction. Results: The extracted DNA obtained indicated high state of integrity and yield. Polymerase chain reaction (PCR) assay using universal panfungal ITS primers successfully amplified the internal transcribed spacer (ITS) region of the extracted DNA samples that led to the identification of six Candida isolates including C. auris. Conclusion: The method is simple, rapid, less costly and environmentally friendly. It is useful in assisting early detection of bloodstream Candida species. The yield of the extracted DNA is adequate to be used as templates for PCR and simple cloning. Summary: Candida DNA isolation is often difficult but critical in early detection of emerging strains, particularly multidrug resistant Candida auris.References: 1. Ali, N., Rampazzo, R. D. C. P., Costa, A. Di. T., & Krieger, M. A. (2017). Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. BioMed Research International, 2017:1 - 13.

Wagner, K., Springer, B., Pires, V. P., & Keller, P. M. (2018). Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture. Scientific Reports, 8(1):1–7.


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