| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Correspondence |
Department of Medical Parasitology and Mycology, School of Public Health and Institute of Public Health Researches, Medical Sciences, Tehran University, Tehran 14155-6446, Iran
Correspondence
Hossein Mirhendi
(mirhendi{at}tums.ac.ir)
Aspergillus fumigatus remains the most frequent cause of invasive aspergillosis; however, other species, including Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans and Aspergillus ustus have been reported to cause human infection (Henry et al., 2000). Rapid and accurate identification of Aspergillus species is necessary for successful clinical management of infection and for epidemiological purposes. Identification of Aspergillus species based on morphological methods requires adequate growth time for evaluation of colony characteristics and microscopic features. A culture time of 5 days or more is generally required for the development of anamorphic forms of Aspergillus. Failure to form conidia on ordinary culture media may require colonies to be further subcultured on specialized media to induce spore formation (Henry et al., 2000; Hinrikson et al., 2005). In addition, morphology tests are usually labour intensive and need expert mycology personnel. Due to these limitations, various molecular approaches have been used for the identification of Aspergillus species isolated from clinical samples, including PCR amplification of targets followed by either fragment length analysis or DNA probe hybridization or sequence analysis (Hinrikson et al., 2005). The aim of this study was to compare the internal transcribed spacer 1 (ITS1)–ITS2 nucleotide sequences of common Aspergillus species and design a PCR-RFLP profile for differentiation of the most medically important Aspergillus species.
Standard strains were provided by Teikyo University Institute of Medical Mycology, Tokyo, Japan. The ITS region of the strains was sequenced using the universal fungal primers ITS1 and ITS4 for reconfirmation of the species, and for using in enzyme selection for RFLP analysis. In addition, 33 clinical and environmental isolates were also used (Table 1
). The isolates were phenotypically identified according to the macroscopic and microscopic morphology of their colonies on Czapek–Dox agar (Difco). All moulds were also cultured on Sabauroud's dextrose agar and incubated at 30 °C for 2 days. Genomic DNA was extracted using single-tube rapid glass-bead disruption (Yamada et al., 2002). Total DNA in the supernatant was precipitated with 2-propanol, resuspended in 100 µl deionized water, and preserved at –20 °C until use. Glass-bead preparation has been used repeatedly for DNA extraction from yeasts (Mirhendi et al., 2006; Yamada et al., 2002). We found that the method is easy to perform and inexpensive tool for rapid extraction of DNA with good recovery.
|
The ITS sequences of Aspergillus species obtained from the GenBank database (Table 2) were aligned and restriction patterns of the sequences for each species were predicted for each of the known restriction enzymes, using DNASIS software (Hitachi Software Engineering). Predicted restriction fragments were compared in order to select those with the best discrimination. Digestion was performed by incubating a 20 µl aliquot of PCR product with 10 U enzyme in a final reaction volume of 25 µl at 37 °C for 2 h, and the digested DNA electrophoresed in 2 % agarose gel according to the manufacturers' instructions.
|
). After analysis of various restriction enzymes, HhaI was selected as the best enzyme for differentiation between five medically important Aspergillus species. The actual size of the PCR products and the predicted size of HhaI-RFLP products for each species are shown in Table 2
. The products of digestion with HhaI in Fig. 1(b), show that the bands generated corresponded to the predicted sizes (Table 2). The restriction enzyme pattern for A. fumigatus is relatively close to A. flavus, yet easily distinguishable. The enzyme was also used on PCR products of 33 clinical and environmental isolates of Aspergillus. No variation was seen in RFLP patterns of each species among different strains (Fig. 2). The results of PCR-RFLPs for identification of the clinical isolates were completely identical to those obtained by conventional morphological methods.
|
|
In order to overcome the limitation of phenotypic methods some genetic targets have been investigated, including the mitochondrial cytochrome b gene (Wang et al., 2001), DNA topoisomerase II gene (TOP II) (Kanbe et al., 2002), and various rDNA regions, such as the small-subunit (18S) and large-subunit (28S) rDNA (Iwen et al., 2002), ITS1 and ITS2 (Henry et al., 2000). Many PCR-based methods have also been used for detecting the target DNA, such as DNA sequence analyses (Henry et al., 2000), DNA probe hybridization (Martinez-Culebras & Ramon, 2007; Meletiadis et al., 2003), PCR enzyme immunoassay (de Aguirre et al., 2004) and single strand conformational polymorphism (Kumeda & Asao, 1996; Rath & Ansorg, 2000). Although there are a few reports of the application of RFLP for identifying some Aspergillus species (Kumeda & Asao, 1996; Martinez-Culebras & Ramon, 2007; Rath & Ansorg, 2000; Somashekar et al., 2004), as far as we know, there is no report about the application of RFLP analyses to identify common pathogenic species of Aspergillus.
In the present study we assessed the utility of the ITS region as a target for an easy-to-perform one-enzyme DNA-based PCR-RFLP method to discriminate the five most common pathogenic Aspergillus species. The profile is based on various ITS sequences belonging to the Aspergillus species deposited in GenBank (Table 1). The ITS region as a DNA target for PCR amplification has some advantages over other molecular targets, e.g. increased sensitivity due to the existence of approximately 100 copies per genome, and sequence variability for distinguishing individual species and stability among strains belonging to a given species. We analysed data and selected an inexpensive restriction enzyme, HhaI, that was capable of the discrimination of the above-mentioned organisms. Our RFLP clearly differentiated the five Aspergillus standard strains. This PCR-restriction enzyme profile is applicable for reference medical microbiology laboratories. Identification and differentiation of the most medically important Aspergillus species from short-term culture using the profile can be a quick, reliable and economical method that provides faster identification than standard culture methods. DNA extraction, PCR and restriction digestion for a dozen samples, can be carried out within 1 working day, compared with morphological procedures that take several days.
ACKNOWLEDGEMENTS
This research has been supported by Tehran University of Medical Sciences and Health Services grant no.240/6866.
REFERENCES
de Aguirre, L., Hurst, S. F., Choi, J. S., Shin, J. H., Hinrikson, H. P. & Morrison, C. J. (2004). Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay. J Clin Microbiol 42, 3495–3504.
Henry, T., Iwen, P. C. & Hinrichs, S. H. (2000). Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J Clin Microbiol 38, 1510–1515.
Hinrikson, H. P., Hurst, S. F., Lott, T. J., Warnock, D. W. & Morrison, C. J. (2005). Assessment of ribosomal large-subunit D1–D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species. J Clin Microbiol 43, 2092–2103.
Iwen, P. C., Hinrichs, S. H. & Rupp, M. E. (2002). Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Med Mycol 40, 87–109.[Medline]
Kanbe, T., Yamaki, K. & Kikuchi, A. (2002). Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene. Microbiol Immunol 46, 841–848.[Medline]
Kumeda, Y. & Asao, T. (1996). Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section flavi. Appl Environ Microbiol 62, 2947–2952.[Abstract]
Martinez-Culebras, P. V. & Ramon, D. (2007). An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine. Int J Food Microbiol 113, 147–153.[CrossRef][Medline]
Meletiadis, J., Melchers, W. J., Meis, J. F., Van Den Hurk, P., Jannes, G. & Verweij, P. E. (2003). Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids. Med Mycol 41, 65–74.[Medline]
Mirhendi, H., Makimura, K., Khoramizadeh, M. & Yamaguchi, H. (2006). A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Nippon Ishinkin Gakkai Zasshi 47, 225–229.[CrossRef][Medline]
Rath, P. M. & Ansorg, R. (2000). Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region. Mycoses 43, 381–386.[CrossRef][Medline]
Somashekar, D., Rati, E. R. & Chandrashekar, A. (2004). PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize. Int J Food Microbiol 93, 101–107.[CrossRef][Medline]
Wang, L., Yokoyama, K., Takahasi, H., Kase, N., Hanya, Y., Yashiro, K., Miyaji, M. & Nishimura, K. (2001). Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene. Int J Food Microbiol 71, 75–86.[CrossRef][Medline]
Yamada, Y., Makimura, K., Mirhendi, H., Ueda, K., Nishiyama, Y., Yamaguchi, H. & Osumi, M. (2002). Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Jpn J Infect Dis 55, 122–125.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. A. Etienne, R. Kano, and S. A. Balajee Development and Validation of a Microsphere-Based Luminex Assay for Rapid Identification of Clinically Relevant Aspergilli J. Clin. Microbiol., April 1, 2009; 47(4): 1096 - 1100. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
