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

Cindy M. Liu, Sergey Kachur, Michael G. Dwan, Alison G. Abraham, Maliha Aziz, Po Ren Hsueh, Yu Tsung Huang, Joseph D. Busch, Louis J. Lamit, Catherine A. Gehring, Paul Keim, Lance B. Price

Research output: Contribution to journalArticle

67 Scopus citations

Abstract

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. 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. We designed FungiQuant, a TaqMan® 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. 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.

Original languageEnglish (US)
JournalUnknown Journal
Volume12
DOIs
StatePublished - 2012
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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    Liu, C. M., Kachur, S., Dwan, M. G., Abraham, A. G., Aziz, M., Hsueh, P. R., Huang, Y. T., Busch, J. D., Lamit, L. J., Gehring, C. A., Keim, P., & Price, L. B. (2012). FungiQuant: a broad-coverage fungal quantitative real-time PCR assay. Unknown Journal, 12. https://doi.org/10.1186/1471-2180-12-255