P497 Candida DNA extraction from positive blood culture bottles

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundMolecular technique of detecting candida from blood is a quick method to identify candidemia. A major limitation of the molecular method is the difficulty associated with breaking fungal cell wall, the DNA extraction step still requires more than half of a working day.The successful extraction of DNA involves effective disruption of cells, denaturation of protein, and nucleoprotein complexes, and inactivation of nucleases such as DNase. The extracted DNA should have low contamination of proteins.ObjectivesTo perform DNA extraction using phenol-chloroform method.2. To speciate candida using PCR-RFLP.Methods Candida DNA is isolated using phenol-chloroform method. The PCR products of different candida species obtained directly from blood samples were subjected to restriction enzyme (MspI).ResultsDNA was extracted from 50 samples, out of which 40 samples were from known positive blood culture bottles and the remaining 10 were known negative for candida. Of the 40 known positive blood culture bottles, 34 produced bands of various molecular weights and all 10 negative cultures did not show bands. Representative amplicon is shown in the gel picture (Figure 1).Out of 40 candida-positive blood culture samples, 7 species have been identified using PCR- RFLP method.ConclusionThe turn-around time can be highly reduced by extracting DNA from positive blood culture bottles and then performing PCR and RFLP for speciation compared with the conventional method.