title: BactQuant: an enhanced broad-coverage bacterial quantitative real-time PCR assay.
abstract: BACKGROUND
  Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay--BactQuant--for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant.
  
  
  METHODS
  The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
  
  
  RESULTS
  A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively.
  
  
  CONCLUSIONS
  The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions.
authors.affiliation: Division of Pathogen Genomics, Translational Genomics Research Institute, 3051 W, Shamrell Blvd,, Suite 106, Flagstaff, AZ 86001, USA.
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Liu
authors.forename: Maliha
authors.initials: M
authors.lastname: Aziz
authors.forename: Sergey
authors.initials: S
authors.lastname: Kachur
authors.forename: Po-Ren
authors.initials: PR
authors.lastname: Hsueh
authors.forename: Yu-Tsung
authors.initials: YT
authors.lastname: Huang
authors.forename: Paul
authors.initials: P
authors.lastname: Keim
authors.forename: Lance B
authors.initials: LB
authors.lastname: Price
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D058491:Bacterial Load / Q000379:methods*; D004275:DNA, Ribosomal / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S