Strain-dependent variation in 18S ribosomal DNA Copy numbers in Aspergillus fumigatus

Abstract

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.

FIG. 1.

Confirmation of arsC copy number in AF293. (A) Priming sites for the two arsC alleles. The Chr1 arsC allele is located on chromosome 1, while the Chr5 arsC allele is located on chromosome 5. Primers are indicated by black arrows; PCR product is indicated by the line connecting the primers. The three NcoI sites (N) (one located within and two flanking the arsC genes), with locations given as base pairs, are indicated within parentheses. Stippled boxes are the arsC open reading frames. The predicted sizes of the fragments after NcoI digestion are indicated below each open reading frame. (B) NcoI digestion of arsC PCR products. Lane 1, uncut Chr1 arsC PCR product; lane 2, NcoI digest of Chr1 arsC; lane 3, mixture of both NcoI digestions; lane 4, NcoI digestion of Chr5 arsC PCR product; lane 5, uncut Chr5 arsC PCR product. Sizes are in base pairs. L, ladder. Ladder sizes are at the right of the gel; fragment sizes are at the left.

FIG. 2.

Amplification plot of AF293 arsC versus FKS1 TaqMan assays. TaqMan assays were performed using an arsC primer-probe combination and FKS1 primer-probe combination. The graph represents a sample plot from duplicate reactions run on aliquots of the same DNA sample. Amplification of the arsC gene is denoted by circles. Amplification of the FKS1 gene is denoted by squares. The C_T value of the arsC line is approximately 21.1 (downward arrow), and the C_T value of FKS1 is approximately 22.1 (upward arrow).

FIG. 3.

Amplification plot of 18S rDNA versus FKS1. An example of copy number determination of 18S rDNA using FKS1 as a single-copy control. The figure is an amplification plot of a TaqMan assay performed using the 18S rDNA primer-probe combination and FKS1 primer-probe combination. Template DNA was taken from the same DNA sample prepared from AF293 and run in duplicate. Note the earlier C_T value of 18S rDNA (circles), which is approximately 18.0 (downward arrow) versus the FKS1 C_T value (squares), which is approximately 23.4 (upward arrow). The lower C_T value for 18S rDNA reflects the greater copy number of the target, since the fluorescence crosses the threshold at a much lower cycle number.