Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Abstract

For PCR‐based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species‐specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species‐specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species‐specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.

Improvements of PCR‐Based Identification Targeting the DNA Topoisomerase II Gene to Determine Major Species of the Opportunistic Fungi Candida and Aspergillus fumigatus

“…In this study, we improved the PCR-based identification targeting the DNA topoisomerase II gene reported previously (8,9), and demonstrated evidences that this improved PCR system method using a single primer set was useful for the detection/identification of the major Candida and Aspergillus species.…”

“…Candida albicans (A9, NUM562 and IAM4966), C. tropicalis (NUM5076, NUM37 and NUM5012), C. parapsilosis (IFO1068, IFO0640, IFO1396, TCH60, NUM644), C. glabrata (ATCC2001, IFO1085 and TIMM1053) and Aspergillus fumigatus (JCM1739, IAM2004 and NUM11009), which have previously been reported elsewhere (8)(9)(10), were used in this study. In addition, C. dubliniensis (16F and 3b), C. guilliermondii (CBS566 and NUM43), C. krusei (IFO0013 and NUM333), C. kefyr (IFO0882 and IFO1065), C. tropicalis I (pk233), C. lusitaniae (JCM5936), Yarroiwa lipolytica (NUM550), Cryptococcus neoformans (IFO0410), A. niger (IFO4066), A. flavus (IAM13836) and A. nidulans (FGSC4) were used to test the specificities of the PCR primers.…”

“…PCR primers designated CDF28 and CDR148, which were previously reported (8,9), were used as common primers for Candida and Aspergillus spp. in the first-round PCR.…”

“…Targeting the DNA Topoisomerase II Gene to Determine Major Species of the Opportunistic Fungi Candida and Aspergillus fumigatus glabrata and A. fumigatus still constitute most fungal infections (8,9,18). For the identification of these species by our previous PCR system, four primer sets were necessary, and these were complicated as a system for the identification of the major opportunistic fungal species.…”

“…During recent years, we have focused on the DNA topoisomerase II genes of pathogenic fungi, and reported not only the characteristics of these genes but also PCR-based identification systems targeting this gene in 10 pathogenic Candida species (12 genotypes) and five Aspergillus species using primer mixes (8)(9)(10)(11)(12). However, a method focusing on the detection/identification of these major fungal species from clinical materials is more constructive under the current circumstances than the method for identification of a broad range of the pathogenic Candida species, since C. albicans, C. parapsilosis, C. tropicalis, C.…”