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Journal of Clinical Microbiology, May 2009, p. 1325-1332, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02073-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus{triangledown}

M. L. Herrera,1 A. C. Vallor,2 J. A. Gelfond,3 T. F. Patterson,2 and B. L. Wickes1,4*

Department of Microbiology and Immunology,1 Department of Medicine,2 Department of Epidemiology and Biostatistics, the University of Texas Health Science Center at San Antonio,3 the San Antonio Cancer Institute, San Antonio, Texas4

Received 27 October 2008/ Returned for modification 4 December 2008/ Accepted 27 February 2009

Enumerating Aspergillus fumigatus CFU can be challenging since CFU determination by plate count can be difficult. CFU determination by quantitative real-time PCR (qPCR), however, is becoming increasingly common and usually relies on detecting one of the subunits of the multicopy rRNA genes. This study was undertaken to determine if ribosomal DNA (rDNA) copy number was constant or variable among different A. fumigatus isolates. FKS1 was used as a single-copy control gene and was validated against single-copy (pyrG and ARG4) and multicopy (arsC) controls. The copy numbers of the 18S rDNA subunit were then determined for a variety of isolates and were found to vary with the strain, from 38 to 91 copies per genome. Investigation of the stability of the 18S rDNA copy number after exposure to a number of different environmental and growth conditions revealed that the copy number was stable, varying less than one copy across all conditions, including in isolates recovered from an animal model. These results suggest that while the ribosomal genes are excellent targets for enumeration by qPCR, the copy number should be determined prior to using them as targets for quantitative analysis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, the University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., Mail Code 7758, San Antonio, TX 78229-3900. Phone: (210) 567-3938. Fax: (210) 567-6612. E-mail: wickes{at}uthscsa.edu

{triangledown} Published ahead of print on 4 March 2009.

Journal of Clinical Microbiology, May 2009, p. 1325-1332, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02073-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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