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Determination of Ribosomal DNA Copy Number and Comparison Among Strains of Coccidioides

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Abstract

The use of PCR-based assays to detect fungi and diagnose fungal infections as well as to monitor fungal organ burden with diseases such as coccidioidomycosis is becoming more common. The target of these assays is frequently one or more of the ribosomal DNA (rDNA) gene subunits. The multicopy nature of this gene affords greater sensitivity over single-copy genes. However, there are few studies reporting the precise number of copies of the rDNA gene per genome in pathogenic fungi. Quantitative PCR was used to determine the number of copies of rDNA as well as CTS1, a single-copy gene, in samples of Coccidioides genomic DNA by the absolute quantification method. Variability of rDNA genome copy number was determined using 13 different Coccidioides isolates and was found to vary between 20 and 146 copies per genome. This suggests that detection of rDNA will likely afford an increased sensitivity of at least 20-fold over single-copy genes. However, estimation of the number of organisms present by quantification of the rDNA cannot be made prior to knowledge of each isolate’s rDNA copy number because of the strain variation.

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References

  1. Fisher MC, Koenig GL, White TJ, Taylor JW. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia. 2002;94:73–84.

    Article  CAS  PubMed  Google Scholar 

  2. Koufoupanou V, Burt A, Taylor JW. Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. Proc Natl Acad Sci. 1997;94:5478–82.

    Article  Google Scholar 

  3. Fisher MC, Koenig GL, White TJ, San-Blas G, Negroni R, Alvarez IG, Wanke B, Taylor JW. Biogeographic range expansion into South America by Coccidioides immitis mirrors new world patterns of human migration. Proc Natl Acad Sci. 2001;98:4558–62.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Tintelnot K, De Hoog GS, Antweiler E, Losert H, Seibold M, Brandt MA, Van Den Ende AH, Fisher MC. Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol. 2007;45:385–93.

    Article  CAS  PubMed  Google Scholar 

  5. Chosewood LC, Wilson DE. Biosafety in microbiological and biomedical laboratories. 5th ed. Washington, DC: US Department of Health and Human service, CDC/NIH, US Government Printing Office; 2009.

  6. Gago S, Buitrago MJ, Clemons KV, Cuenca-Estrella M, Mirels LF, Stevens DA. Development and validation of a quantitative real-time PCR assay for the early diagnosis of coccidioidomycosis. Diagn Microbiol Infect Dis. 2014;79:214–21.

    Article  CAS  PubMed  Google Scholar 

  7. de Macedo RCL, Rosado AS, da Mota FF, Cavalcante MAS, Eulalio KD, Filho A, Martins LMS, Lazera MS, Wanke B. Molecular identification of Coccidioides spp. in soil samples from Brazil. BMC Microbiol. 2011;11:108.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Lauer A, Baal JDH, Baal JCH, Verma M, Chen JM. Detection of Coccidioides immitis in Kern County, California, by multiplex PCR. Mycologia. 2012;104:62–9.

    Article  CAS  PubMed  Google Scholar 

  9. Baptista-Rosas RC, Catalan-Dibene J, Romero-Olivares AL, Hinojosa A, Cavazos T, Riquelme M. Molecular detection of Coccidioides spp. from environmental samples in Baja California: linking valley fever to soil and climate conditions. Fungal Ecol. 2012;5:177–90.

    Article  Google Scholar 

  10. Diab S, Johnson SM, Garcia J, Carlson EL, Pappagianis D, Smith J, Uzal FA. Case report: abortion and disseminated infection by Coccidioides posadasii in an alpaca (Vicugna pacos) fetus in Southern California. Med Mycol Case Rep. 2013;2:159–62.

    Article  PubMed Central  PubMed  Google Scholar 

  11. Johnson SM, Carlson EL, Fisher FS, Pappagianis D. Demonstration of Coccidioides immitis and Coccidioides posadasii DNA in soil samples collected from Dinosaur National Monument, Utah. Med Mycol. 2014;52:610–7.

    Article  PubMed  Google Scholar 

  12. Canteros CE, Toranzo A, Suarez-Alvarez R, Davel G, Castanon-Olivares LR, Napoli J. Genetic characterization of the fungus involved in the first case of coccidioidomycosis described by Alejandro Posadas in 1892. Medicina (B Aires). 2009;69:215–20.

    Google Scholar 

  13. Borsuk PA, Nagiec MM, Stepien PP, Bartnik E. Organization of the ribosomal RNA gene cluster in Aspergillus nidulans. Gene. 1982;17:147–52.

    Article  CAS  PubMed  Google Scholar 

  14. Garber RC, Turgeon BG, Selker EU, Yoder OC. Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus. Curr Genet. 1988;14:573–82.

    Article  CAS  PubMed  Google Scholar 

  15. Herrera ML, Vallor AC, Gelfond JA, Patterson TF, Wickes BL. Strain-dependent variation in 18S ribosomal DNA copy numbers in Aspergillus fumigatus. J Clin Microbiol. 2009;47:1325–32.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Longo AV, Rodriquez D, da Silva Leite D, Toledo LF, Almeralla CM, Burrowes PA, Zamudio KR. ITS1 copy number varies among Batrachochytrium dendrobatidis strains: implication for qPCR estimates of infection intensity from field-collected amphibian skin swabs. PLoS One. 2013;8:e59499.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Friedman L, Smith CE, Roessler WG, Berman RJ. The virulence and infectivity of twenty-seven strains of Coccidioides immitis. Am J Hyg. 1956;64:198–210.

  18. Friedman L, Smith CE. The comparison of four strains of Coccidioides immitis with diverse histories. Mycopathol Mycol Appl. 1957;8:47–53.

    Article  CAS  PubMed  Google Scholar 

  19. Zimmermann CR, Snedker CJ, Pappagianis D. Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms. J Clin Microbiol. 1994;32:3040–2.

    CAS  PubMed Central  PubMed  Google Scholar 

  20. Walch HA, Walch RK. Studies with induced mutants of Coccidioides immitis. In: Ajello L, editor. Coccidioidomycosis. Tucson: University of Arizona Press; 1967. p. 339–47.

    Google Scholar 

  21. Walch HA, Kalvoda A. Immunization of mice with induced mutants of Coccidioides immitis. I. Characterization of mutants and preliminary studies of their use as viable vaccines. Sabouraudia. 1971;9:173–84.

    Article  CAS  PubMed  Google Scholar 

  22. Davis BL, Smith RT, Smith CE. An epidemic of coccidioidal infection (coccidioidomycosis). J Am Med Assoc. 1942;118:1182–6.

    Article  Google Scholar 

  23. Zimmermann CR, Johnson SM, Martens GW, White AG, Pappagianis D. Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis. Infect Immun. 1996;64:4967–75.

    CAS  PubMed Central  PubMed  Google Scholar 

  24. Martin KJ, Rygiewicz PT. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol. 2005;5:28.

    Article  PubMed Central  PubMed  Google Scholar 

  25. Reichard U, Hung CY, Thomas PW, Cole GT. Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis. Infect Immun. 2000;68:5830–8.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

  27. Applied Biosystems. The comparative CT method (ΔΔCT method). In: Guide to performing relative quantitation of gene expression using real-time quantitative PCR. Foster City, CA: Applied Biosystems; 2008. p. 52–3.

  28. Candela M, Vitali B, Matteuzzi D, Brigidi P. Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR. Lett Appl Microbiol. 2004;38:229–32.

    Article  CAS  PubMed  Google Scholar 

  29. Lee C, Lee S, Shin SG, Hwang S. Real-time PCR determination of rRNA copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotechnol. 2008;78:371–6.

    Article  CAS  PubMed  Google Scholar 

  30. Pappagianis D. Current status of serologic studies in coccidioidomycosis. Curr Fungal Infect Rep. 2007;1:129–34.

    Google Scholar 

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Acknowledgments

We acknowledge the assistance of the Real-Time PCR Core Facility at UC Davis with design of qPCR primers and probes as well as with the performance of qPCR assays.

Conflict of interest

The authors report no conflict of interest.

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Author notes

  1. Erin L. Carlson

    Present address: Department of Psychiatry and Behavior Sciences, School of Medicine, University of California, Davis, 2805 50th Street, Sacramento, CA, 95817, USA

Authors and Affiliations

  1. Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, 1 Shields Avenue, Davis, CA, 95616, USA

    Suzanne M. Johnson, Erin L. Carlson & Demosthenes Pappagianis

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  1. Suzanne M. Johnson
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  3. Demosthenes Pappagianis
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Correspondence to Suzanne M. Johnson.

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Johnson, S.M., Carlson, E.L. & Pappagianis, D. Determination of Ribosomal DNA Copy Number and Comparison Among Strains of Coccidioides . Mycopathologia 179, 45–51 (2015). https://doi.org/10.1007/s11046-014-9820-y

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  • DOI: https://doi.org/10.1007/s11046-014-9820-y

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