Development and validation of Loop-Mediated Isothermal Amplification (LAMP) detection limit using blood matrix spike for Candida glabrata detection

Abstract

Background: Rapid and accurate identification of yeast responsible for invasive candidiasis (IC) allows for focused and early initiation of therapy for good clinical outcome. Candida glabrata is increasingly important, and the diagnostic sensitivity of microbial culture method “gold standard” is low and could miss up to 50% of the IC patients. To develop and validate LAMP technique detection limit in comparison to microbial culture and PCR techniques using blood matrix spike for C. glabrata detection. Methods and materials: Blood spiking experiment was conducted using healthy blood sample. Candida glabrata ATCC 2001 reference strain colonies were re-suspended in phosphate-buffered saline (PBS) buffer to 0.5 McFarland standard. Ten-fold serial dilutions of yeast suspensions in 1x PBS was made from 106 to 101 cfu/mL concentrations. A 100 μL from each dilution was plated onto SDA and the resulting number of colonies was established. A 25 μL of each dilution was spiked in 225 μL of blood sample (1:10 dilution). The C. glabrata DNA was extracted from spiked blood using Qiagen yeast DNA extraction kit. The developed LAMP assay detects C. glabrata using a primer set (three pairs) (FIP/BIP, F3/B3 and LF/LB) that recognized eight distinct regions on the target internal transcribed spacer (ITS) gene, while, ITS1/4 universal primers were used in PCR analysis. The cfu/mL detection limit of the LAMP was compared to culture and PCR techniques. Results: The LAMP assay analysis of DNA from spiked blood, indicates detection limit of 101 cfu/mL (4.03 x 101 cells/25 μL) using gel electrophoresis analysis. The PCR detection limit was 105 cfu/mL (4.03 x 104 cells/25 μL) and the culture detection limit was 104 cfu/mL (1.61 x 106 cells/mL). Conclusion: The LAMP assay indicates high sensitivity of C. glabrata ITS detection from spiked blood compared to PCR and therefore, a good diagnostic tool for molecular analysis.