title: FungiQuant: a broad-coverage fungal quantitative real-time PCR assay.
abstract: 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® 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.
authors.affiliation: Division of Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, AZ 86011, USA. cindy.liu@nau.edu
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Liu
authors.forename: Sergey
authors.initials: S
authors.lastname: Kachur
authors.forename: Michael G
authors.initials: MG
authors.lastname: Dwan
authors.forename: Alison G
authors.initials: AG
authors.lastname: Abraham
authors.forename: Maliha
authors.initials: M
authors.lastname: Aziz
authors.forename: Po-Ren
authors.initials: PR
authors.lastname: Hsueh
authors.forename: Yu-Tsung
authors.initials: YT
authors.lastname: Huang
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Busch
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Lamit
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Gehring
authors.forename: Paul
authors.initials: P
authors.lastname: Keim
authors.forename: Lance B
authors.initials: LB
authors.lastname: Price
mesh_terms: D015169:Colony Count, Microbial / Q000379:methods*; D005658:Fungi / Q000302:isolation & purification; D020459:Genes, rRNA; D006801:Humans; D054892:Metagenome; D012331:RNA, Fungal / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S