FungiQuant: a broad-coverage fungal quantitative real-time PCR assay

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Accurate fungal load quantification is essential for understanding fungal communities.
• Current methods for quantifying fungi in the human microbiome have limitations.
• There is a need for novel, broad-coverage techniques for fungal quantification.

Purpose of the Study:

• To develop and validate a novel quantitative PCR (qPCR) assay for broad-coverage fungal quantification.
• To ensure the assay can accurately distinguish fungal DNA from human DNA.
• To meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.

Main Methods:

• Designed a TaqMan® qPCR assay (FungiQuant) targeting the fungal 18S rRNA gene.
• Analyzed 2,085 18S rRNA gene sequences for assay design and validated against 4,968 sequences.
• Quantified plasmid standards and validated assay performance using MIQE guidelines.

Main Results:

• FungiQuant demonstrated perfect sequence match to 90.0% of analyzed fungal species.
• The assay showed 100% sensitivity with high amplification efficiency and reproducibility.
• FungiQuant achieved a limit of quantification of 25 copies and a limit of detection of 5 copies, distinguishing fungal from human DNA.

Conclusions:

• FungiQuant is a robust qPCR assay for accurate fungal quantification and detection.
• The assay offers comprehensive coverage across diverse fungi.
• FungiQuant effectively delineates true fungal detection from non-target human DNA in microbiome samples.