期刊
BMC MICROBIOLOGY
卷 12, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/1471-2180-12-255
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资金
- National Institutes of Health [R01AI087409-01A1, R15DE021194-01]
- Department of Defense [W81XWH1010870]
- TGen Foundation
- Northern Arizona University Technology and Research Initiative Fund (TRIF) fund
- Cowden Endowment in Microbiology at Northern Arizona University
- U.S. Department of Defense (DOD) [W81XWH1010870] Funding Source: U.S. Department of Defense (DOD)
Background: Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods: We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results: We designed FungiQuant, a TaqMan (R) qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r(2)-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter-and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. Conclusions: FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.
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