Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR

Abstract

A commercially available repetitive-sequence-based PCR (rep-PCR) DNA fingerprinting assay adapted to an automated format, the DiversiLab system, enables rapid microbial identification and strain typing. We explored the performance of the DiversiLab system as a molecular typing tool for 69 Aspergillus isolates (38 A. fumigatus, 15 A. flavus, and 16 A. terreus isolates) had been previously characterized by morphological analysis. Initially, 27 Aspergillus isolates (10 A. fumigatus, 9 A. flavus, and 8 A. terreus isolates) were used as controls to create a rep-PCR-based DNA fingerprint library with the DiversiLab software. Then, 42 blinded Aspergillus isolates were typed using the system. The rep-PCR-based profile revealed 98% concordance with morphology-based identification. rep-PCR-based DNA fingerprints were reproducible and were consistent for DNA from both hyphae and conidia. DiversiLab dendrogram reports correctly identified all A. fumigatus (n = 28), A. terreus (n = 8), and A. flavus (n = 6) isolates in the 42 blinded Aspergillus isolates. rep-PCR-based identification of all isolates was 100% in agreement with the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) sequence-based identification of the respective isolates. Additionally, the DiversiLab system could demonstrate strain-level differentiation of A. flavus and A. terreus. Automated rep-PCR may be a time-efficient, effective, easy-to-use, novel genotyping tool for identifying and determining the strain relatedness of fungi. This system may be useful for epidemiological studies, molecular typing, and surveillance of Aspergillus species.

FIG. 1.

Overview of automated rep-PCR and the DiversiLab system. (A) rep-PCR primers bind to many specific repetitive sequences interspersed throughout the genome and amplify various fragments of different lengths. (B) A microfluidics chip separates amplified fragments based on size and charge. The fluorescence intensity and migration time are used to generate electropherograms from each DNA sample. (C) A sample DiversiLab report showing data analysis of the samples, gel-like images, and the dendrogram.

FIG. 2.

DiversiLab system-generated dendrogram and scatter plot of the 27 known Aspergillus isolates. The dendrogram (A) and scatter plot (B) show species clustering. A rep-PCR-based DNA fingerprinting library was simultaneously generated for comparison purposes. The horizontal bar at the bottom left of the dendrogram indicates the percent similarity coefficient within the species. The scatter plot shows a cluster of strains for each Aspergillus species, as indicated by the circles. Spacing between grid lines indicates increments of 5% similarity based on the proximity matrix results as represented using multidimensional scaling. All species assignments are based on morphological identification.

FIG. 3.

Reproducibility and stability of the rep-PCR-based fingerprints of Aspergillus DNA. (A) DNA from each of 27 Aspergillus isolates was extracted and amplified in triplicate. The dendrogram shows rep-PCR profiles in triplicate for two strains of each of the three Aspergillus species. (B) DNA from hyphae and conidia of each of the 42 blinded Aspergillus isolates was extracted and amplified in duplicate. The dendrogram shows rep-PCR profiles of DNA from hyphae (h) and conidia (c) of two strains of each of the three Aspergillus species. The horizontal bar at the bottom left of each of the dendrograms indicates the percent similarity coefficient within the strains.

FIG. 4.

Comparison between rep-PCR-based DNA fingerprint and ITS1-5.8S-ITS2 region sequence analysis of 69 Aspergillus isolates. DNA from each of the 69 (27 known and 42 blinded) Aspergillus isolates was extracted, amplified by rep-PCR, and sequenced. (A) rep-PCR dendrogram shows the rep-PCR-based DNA profiles of 27 known and 17 of the 42 blinded (shown in circle) Aspergillus isolates. (B) ITS1-5.8S-ITS2 sequence-based phylogenetic tree of the same Aspergillus isolates. The horizontal bar at the bottom left of the dendrogram and the phylogenetic tree indicate the percent similarity coefficient within the strains.

FIG. 5.

rep-PCR-based strain discrimination of A. terreus. DNA from each of 16 strains of A. terreus was extracted and amplified via rep-PCR. The horizontal bar at the bottom left of the dendrogram indicates the percent similarity coefficient within the strains. CL, colonizing strain; IA, invasive strains.