FungiQuant: a Broad-coverage Fungal Quantitative Real-time PCR Assay

Overview

Journal BMC Microbiol

Publisher Biomed Central

Specialty Microbiology

Date 2012 Nov 10

PMID 23136846

Citations 89

Authors

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

Affiliations

Soon will be listed here.

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.

Citing Articles

Impact of Saharan Dust and Gene Variants on Bacterial/Fungal Balance in Asthma Patients.

Escuela-Escobar A, Perez-Garcia J, Martin-Gonzalez E, Gonzalez Martin C, Hernandez-Perez J, Gonzalez Perez R Int J Mol Sci. 2025; 26(5).

PMID: 40076778 PMC: 11899813. DOI: 10.3390/ijms26052158.

Genomic interrogation of commercial infant probiotic products for label accuracy.

DeVette C, Sugino K, Wilson A, Fuller K, Friedman J, Burge K Pediatr Res. 2025; .

PMID: 39856233 DOI: 10.1038/s41390-025-03831-6.

Detection of fungal sequences in human brain: rDNA locus amplification and deep sequencing.

Leitao R, Wan I, Chown H, Williams T, Fisher M, Rhodes J Sci Rep. 2024; 14(1):31790.

PMID: 39738312 PMC: 11685392. DOI: 10.1038/s41598-024-82840-7.

Evaluation of inter- and intra-variability in gut health markers in healthy adults using an optimised faecal sampling and processing method.

Kruger K, Myeonghyun Y, van der Wielen N, Kok D, Hooiveld G, Keshtkar S Sci Rep. 2024; 14(1):24580.

PMID: 39427011 PMC: 11490648. DOI: 10.1038/s41598-024-75477-z.

Disruption of millipede-gut microbiota in E. pulchripes and G. connexa highlights the limited role of litter fermentation and the importance of litter-associated microbes for nutrition.

Nweze J, Gupta S, Salcher M, Sustr V, Horvathova T, Angel R Commun Biol. 2024; 7(1):1204.

PMID: 39342029 PMC: 11438867. DOI: 10.1038/s42003-024-06821-2.

References

Verdaguer V, Walsh T, Hope W, Cortez K . Galactomannan antigen detection in the diagnosis of invasive aspergillosis. Expert Rev Mol Diagn. 2006; 7(1):21-32. DOI: 10.1586/14737159.7.1.21. View

Amend A, Seifert K, Bruns T . Quantifying microbial communities with 454 pyrosequencing: does read abundance count?. Mol Ecol. 2010; 19(24):5555-65. DOI: 10.1111/j.1365-294X.2010.04898.x. View

Oz Y, Kiraz N . Diagnostic methods for fungal infections in pediatric patients: microbiological, serological and molecular methods. Expert Rev Anti Infect Ther. 2011; 9(3):289-98. DOI: 10.1586/eri.10.168. View

Olsson P, Larsson L, Bago B, Wallander H, Van Aarle I . Ergosterol and fatty acids for biomass estimation of mycorrhizal fungi. New Phytol. 2021; 159(1):7-10. DOI: 10.1046/j.1469-8137.2003.00810.x. View

Ide S, Miyazaki T, Maki H, Kobayashi T . Abundance of ribosomal RNA gene copies maintains genome integrity. Science. 2010; 327(5966):693-6. DOI: 10.1126/science.1179044. View

. Finishing the euchromatic sequence of the human genome. Nature. 2004; 431(7011):931-45. DOI: 10.1038/nature03001. View

Herrera M, Vallor A, Gelfond J, Patterson T, Wickes B . Strain-dependent variation in 18S ribosomal DNA Copy numbers in Aspergillus fumigatus. J Clin Microbiol. 2009; 47(5):1325-32. PMC: 2681831. DOI: 10.1128/JCM.02073-08. View

Geha D, Roberts G . Laboratory detection of fungemia. Clin Lab Med. 1994; 14(1):83-97. View

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

10.

Dickie I, FitzJohn R . Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza. 2007; 17(4):259-270. DOI: 10.1007/s00572-007-0129-2. View

11.

Davey M, Nybakken L, Kauserud H, Ohlson M . Fungal biomass associated with the phyllosphere of bryophytes and vascular plants. Mycol Res. 2009; 113(Pt 11):1254-60. DOI: 10.1016/j.mycres.2009.08.001. View

12.

McGONIGLE T, Miller M, Evans D, Fairchild G, Swan J . A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol. 2021; 115(3):495-501. DOI: 10.1111/j.1469-8137.1990.tb00476.x. View

13.

Morgulis A, Coulouris G, Raytselis Y, Madden T, Agarwala R, Schaffer A . Database indexing for production MegaBLAST searches. Bioinformatics. 2008; 24(16):1757-64. PMC: 2696921. DOI: 10.1093/bioinformatics/btn322. View

14.

Mennink-Kersten M, Donnelly J, Verweij P . Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis. 2004; 4(6):349-57. DOI: 10.1016/S1473-3099(04)01045-X. View

15.

Park H, Ha M, Park S, Kim M, Kim B, Kim W . Characterization of the fungal microbiota (mycobiome) in healthy and dandruff-afflicted human scalps. PLoS One. 2012; 7(2):e32847. PMC: 3290624. DOI: 10.1371/journal.pone.0032847. View

16.

Kibbe W . OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 2007; 35(Web Server issue):W43-6. PMC: 1933198. DOI: 10.1093/nar/gkm234. View

17.

Ostrosky-Zeichner L . Invasive mycoses: diagnostic challenges. Am J Med. 2011; 125(1 Suppl):S14-24. DOI: 10.1016/j.amjmed.2011.10.008. View

18.

Fisher M, Henk D, Briggs C, Brownstein J, Madoff L, McCraw S . Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012; 484(7393):186-94. PMC: 3821985. DOI: 10.1038/nature10947. View

19.

Ghannoum M, Jurevic R, Mukherjee P, Cui F, Sikaroodi M, Naqvi A . Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 2010; 6(1):e1000713. PMC: 2795202. DOI: 10.1371/journal.ppat.1000713. View

20.

Kobayashi T . Regulation of ribosomal RNA gene copy number and its role in modulating genome integrity and evolutionary adaptability in yeast. Cell Mol Life Sci. 2011; 68(8):1395-403. PMC: 3064901. DOI: 10.1007/s00018-010-0613-2. View