BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

Cindy M. Liu, Maliha Aziz, Sergey Kachur, Po Ren Hsueh, Yu Tsung Huang, Paul S Keim, Lance B. Price

Research output: Contribution to journalArticle

61 Citations (Scopus)

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(R) 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.

Original languageEnglish (US)
Pages (from-to)56
Number of pages1
JournalBMC Microbiology
DOIs
StateAccepted/In press - Apr 17 2012
Externally publishedYes

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rRNA Genes
Real-Time Polymerase Chain Reaction
Bacterial Load
Computer Simulation
Gene Dosage
Publications
Plasmids
Databases
Guidelines
Bacteria

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Liu, C. M., Aziz, M., Kachur, S., Hsueh, P. R., Huang, Y. T., Keim, P. S., & Price, L. B. (Accepted/In press). BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay. BMC Microbiology, 56. https://doi.org/10.1186/1471-2180-12-56

BactQuant : An enhanced broad-coverage bacterial quantitative real-time PCR assay. / Liu, Cindy M.; Aziz, Maliha; Kachur, Sergey; Hsueh, Po Ren; Huang, Yu Tsung; Keim, Paul S; Price, Lance B.

In: BMC Microbiology, 17.04.2012, p. 56.

Research output: Contribution to journalArticle

Liu, Cindy M. ; Aziz, Maliha ; Kachur, Sergey ; Hsueh, Po Ren ; Huang, Yu Tsung ; Keim, Paul S ; Price, Lance B. / BactQuant : An enhanced broad-coverage bacterial quantitative real-time PCR assay. In: BMC Microbiology. 2012 ; pp. 56.
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