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 S Keim, Lance B. Price

Research output: Contribution to journalArticle

52 Citations (Scopus)

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(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 r2-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.

Original languageEnglish (US)
Pages (from-to)255
Number of pages1
JournalBMC Microbiology
DOIs
StateAccepted/In press - Nov 8 2012
Externally publishedYes

Fingerprint

Fungal Structures
rRNA Genes
Real-Time Polymerase Chain Reaction
Fungal DNA
Microbiota
DNA
Computer Simulation
Limit of Detection
Publications
Plasmids
Fungi
Databases
Guidelines

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Liu, C. M., Kachur, S., Dwan, M. G., Abraham, A. G., Aziz, M., Hsueh, P. R., ... Price, L. B. (Accepted/In press). FungiQuant: A broad-coverage fungal quantitative real-time PCR assay. BMC Microbiology, 255. https://doi.org/10.1186/1471-2180-12-255

FungiQuant : A broad-coverage fungal quantitative real-time PCR assay. / Liu, Cindy M.; Kachur, Sergey; Dwan, Michael G.; Abraham, Alison G.; Aziz, Maliha; Hsueh, Po Ren; Huang, Yu Tsung; Busch, Joseph D.; Lamit, Louis J.; Gehring, Catherine A; Keim, Paul S; Price, Lance B.

In: BMC Microbiology, 08.11.2012, p. 255.

Research output: Contribution to journalArticle

Liu, CM, Kachur, S, Dwan, MG, Abraham, AG, Aziz, M, Hsueh, PR, Huang, YT, Busch, JD, Lamit, LJ, Gehring, CA, Keim, PS & Price, LB 2012, 'FungiQuant: A broad-coverage fungal quantitative real-time PCR assay', BMC Microbiology, pp. 255. https://doi.org/10.1186/1471-2180-12-255
Liu CM, Kachur S, Dwan MG, Abraham AG, Aziz M, Hsueh PR et al. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay. BMC Microbiology. 2012 Nov 8;255. https://doi.org/10.1186/1471-2180-12-255
Liu, Cindy M. ; Kachur, Sergey ; Dwan, Michael G. ; Abraham, Alison G. ; Aziz, Maliha ; Hsueh, Po Ren ; Huang, Yu Tsung ; Busch, Joseph D. ; Lamit, Louis J. ; Gehring, Catherine A ; Keim, Paul S ; Price, Lance B. / FungiQuant : A broad-coverage fungal quantitative real-time PCR assay. In: BMC Microbiology. 2012 ; pp. 255.
@article{de750c77934048b18065411c2bc82ced,
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(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 r2-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.",
author = "Liu, {Cindy M.} and Sergey Kachur and Dwan, {Michael G.} and Abraham, {Alison G.} and Maliha Aziz and Hsueh, {Po Ren} and Huang, {Yu Tsung} and Busch, {Joseph D.} and Lamit, {Louis J.} and Gehring, {Catherine A} and Keim, {Paul S} and Price, {Lance B.}",
year = "2012",
month = "11",
day = "8",
doi = "10.1186/1471-2180-12-255",
language = "English (US)",
pages = "255",
journal = "BMC Microbiology",
issn = "1471-2180",
publisher = "BioMed Central",

}

TY - JOUR

T1 - FungiQuant

T2 - A broad-coverage fungal quantitative real-time PCR assay

AU - Liu, Cindy M.

AU - Kachur, Sergey

AU - Dwan, Michael G.

AU - Abraham, Alison G.

AU - Aziz, Maliha

AU - Hsueh, Po Ren

AU - Huang, Yu Tsung

AU - Busch, Joseph D.

AU - Lamit, Louis J.

AU - Gehring, Catherine A

AU - Keim, Paul S

AU - Price, Lance B.

PY - 2012/11/8

Y1 - 2012/11/8

N2 - 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 r2-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.

AB - 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 r2-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.

UR - http://www.scopus.com/inward/record.url?scp=84868459501&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84868459501&partnerID=8YFLogxK

U2 - 10.1186/1471-2180-12-255

DO - 10.1186/1471-2180-12-255

M3 - Article

C2 - 23136846

AN - SCOPUS:84868459501

SP - 255

JO - BMC Microbiology

JF - BMC Microbiology

SN - 1471-2180

ER -