DOI: 10.48802/owey.g54a-kk20
title: Quantification of the intestinal C. albicans carriage and fungal burden in 695 healthy subjects from the Milieu Intérieur cohort

description: The Excel file is composed of 3 columns. The first one corresponds to the subject ID. The second column correspond to the fungal load measured in each subjects and the third correspond to the quantification of C. albicans levels. The fungal load was obtained by dividing the concentration of fungal DNA, measured by 18S Panfungal qPCR in DNA extracted from the fecal samples of each subjects,divided by the total concentration of DNA measured in each samples. C. albicans carriage, reported here in ng/g of fecal sample, was estimated by specific C. albicans qPCR.
description: 1) Fecal DNA extraction:
Eight hundred and twenty one fecal samples from Milieu Intérieur were available for this study and were thus extracted with the following method. For each sample, 100-250 mg of stool was processed following the repeated bead beating plus column method described by Yu and Morrison, 2004 54 except that a Bullet Blender (NextAdvance, Troy, NY, USA) was used instead of a Mini-BeadbeaterTM. DNA samples were eluted in 100 μL of double distilled H2O. Total fecal DNA levels were measured by Qubit (Invitrogen, USA) using the dsDNA Broad Range Kit (Invitrogen, USA). Only DNA extract with a total DNA concentration above 50 ng/μL were retained for analysis, leading to a total of 695 samples that were further analyzed.

2) Quantitative PCR for detection of total fungal load in DNA from human fecal samples:
Fungal DNA was quantified by TaqMan qPCR as described by Liu et al. 2012 using a double dye MGB 5' 6-FAM-labelled probe (Eurogentec, Belgium). All reactions were performed on a CFX96 Real-Time PCR system (BioRad, USA) with the following conditions: 2 min at 50°C, 10 min at 95°C, 15 s at 95°C and 1 min at 60°C, the last two steps repeated for 45 cycles. All samples were tested in two independent rounds, each time in duplicates. The presence of qPCR inhibitors was determined with a duplex internal control - C. albicans qPCR assay, as described below. The fungal load was estimated by dividing the fungal DNA concentration by the total DNA concentration of the sample 56, obtained by Qubit Broad Range protocol.

3) Duplex quantitative PCR for detection of C. albicans DNA in DNA from human fecal samples and internal amplification control:
Ten μL of a 1:10 dilution of the extracted total fecal DNA were used as a template for TaqMan qPCR analysis, using C. albicans probe and primers described by Guiver et al, 2001 57 in combination with the Cy®5-QXL®670 Probe system of the Universal Exogenous qPCR Positive Control for TaqMan® Assay (Eurogentec, Belgium), in order to identify samples with qPCR inhibitors.C. albicans primers and probes were used at 100nM and 400nM concentrations, respectively. All reactions were performed on a CFX96 Real-Time PCR system (BioRad, USA) with the following conditions: 2 min at 50°C, 10 min at 95°C, 15 s at 95°C and 1 min at 60°C, the last two steps repeated for 45 cycles. All samples were quantified in two independent rounds, each time in duplicates. Samples with qPCR inhibitors at a 1:10 dilution were diluted to 1:100 and submitted to a new round of qPCR. A detection threshold of 10-8 ng/μL of DNA was used for this assay.