P452 Estimation of the detection limit of extracted Candida DNA from spiked EDTA blood sample

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundCandidemia caused by genus Candida, are opportunistic pathogens with life-threatening infection in immunocompromised individuals with a mortality rate being 40%–54% in patients. Blood culture is not a reliable and rapid method for the diagnosis of candidemia as its sensitivity is as low as 50%. Molecular diagnosis is an alternative to conventional culture for early detection of candidemia. An appropriate DNA isolation technique is needed to obtain high purity DNA from blood specimens and improves the sensitivity of the polymerase chain reaction (PCR). In this study, two different methods were used to extract Candida DNA from spiked EDTA blood specimens, the In-house phenol-chloroform and potassium acetate method.ObjectivesDNA extraction from the spiked blood samplesQuantification of the Candida DNA extracted from the spiked sampleComparison of the extracted DNA by phenol-chloroform and potassium acetate methodMethodsA total of 5 ml of EDTA blood samples from healthy volunteers were spiked with 104-108 Candida albicans cells (ATCC 90028) to determine the detection limit of our extraction method. DNA was extracted from whole blood using phenol-chloroform and the potassium acetate methods which involves pretreatment with erythrocytes, leukocytes and fungal lysis buffer. DNA from C. albicans were amplified using ITS1 and ITS4 based primers. PCR products were visualized by agarose gel electrophoresis.ResultsThe spiked Candida cells taken were from 104-108 yeast cells. The total extracted DNA by phenol chloroform extraction method ranged from 18.29 to 51.64 ng/μl respectively. Whereas the total extracted DNA from potassium acetate extraction method ranged from 15.58 to 32.30 ng/μl respectively. The presence of a 535 base pair (bp) product was considered positive (Figs. 1 and 2). The lowest detection limit of PCR has been determined as 106-108 Candida cells for our spiked samples.ConclusionIn our study, till date, the extracted quantity of DNA was found to be higher by phenol-chloroform method as compared to the potassium acetate method. Greater numbers and processing are required to obtain conclusive data.