Novel Approach to Designing Primers for Identification and Distinction of the Human Pathogenic Fungi Coccidioides immitis and Coccidioides p
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微生物临床杂志 2006年第5期
Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo 162-8640
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-8673, Japan
ABSTRACT
We developed a pair of primers that specifically identifies Coccidioides species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing Coccidioides immitis and Coccidioides posadasii by simply comparing the amplicon sizes on an agarose gel.
TEXT
Coccidioidomycosis, a fungal respiratory disease of humans caused by Coccidioides species, is endemic to arid areas of the Americas. Two species of Coccidioides are now recognized, whereas until recently, coccidioidomycosis was attributed to only one species, Coccidioides immitis (4). Coccidioides posadasii, formerly known as the non-California C. immitis strain, is found mainly in Texas, Arizona, and regions of endemicity outside of the United States, whereas C. immitis is found primarily in the Central Valley of California. These two species can be divided based on single-nucleotide polymorphisms and the size of microsatellites (3, 4), although the colony morphologies, growth rates, and clinical presentations are almost identical. Because identification of Coccidioides spp. carries with it a great deal of risk, molecular diagnosis without culturing has long been expected. Many researchers have explored nucleic acid detection for the diagnosis of coccidioidomycosis (2, 7-9, 11, 12).
For isolates of C. immitis and C. posadasii, listed in Table 1, the same DNA samples previously described by Sano et al. (10) were used. For non-Coccidioides fungal isolates, listed in Table 2, DNA isolation was performed using a DEXPAT DNA extraction kit (Takara Bio Inc., Japan). PCR was performed with approximately 10 ng of extracted DNA in a 20-μl reaction volume consisting of LA Taq buffer II (Mg2+ plus) (Takara Bio Inc.), 200 μM deoxynucleoside triphosphates, 2.5 U of ExTaq DNA polymerase (Takara Bio Inc.), and 10 pmol of each primer. One cycle at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 45 s with a final extension step at 72°C for 3 min was performed in a PTC-200 DNA Engine thermal cycler (Bio-Rad). The amplified DNA must be handled carefully in order to avoid amplicon contamination. The universal fungal primers ITS1 and ITS4 were used in all DNA samples to verify the efficiency of the test and to ensure that there was no PCR inhibition in the DNA samples (8). Ten microliters of each PCR product was electrophoresed through 2% agarose gel and visualized with a UV light after ethidium bromide or SYBR Safe (Invitrogen) staining. The PCR products were purified from gel with a NucleoSpin Extract II kit (Macherey-Nagel, Germany). Nucleotide sequences were determined using a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) and an ABI 3130 genetic analyzer (Applied Biosystems).
Empirically, the construction of diagnostic primers is based on a nucleotide sequence encoding conserved enzymes or rRNA. However, we left the matter to chance: we repeated the primer construction based on randomly selected regions and verification by actual PCR experiments. In step 1, we obtained a text file containing the C. immitis genome sequence (http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis/). A nucleotide sequence corresponding to the 240- to 720-bp length was randomly selected from the genome database file. We designed 20-mer forward and reverse primers, which were expected to amplify the randomly selected region. In step 2, we examined whether these two primers could amplify DNA fragments of the anticipated size from Coccidioides spp. In step 3, we tested whether the primers that could amplify Coccidioides DNA in step 2 were unable to amplify Candida albicans and Aspergillus fumigatus DNA. By repeating these steps on 64 selected regions, we nominated one pair of primers for more detailed examination. Since this experimental primer design led to our successful product, mentioned below, this strategy should be a powerful method for the development of diagnostic primers.
The selected primers were Coi9-1F (5'-TACGGTGTAATCCCGATACA-3') and Coi9-1R (5'-GGTCTGAATGATCTGACGCA-3'). The selected primer set was constructed to amplify a 720-bp amplicon that corresponds to nucleotide position 660313 to 661032 of C. immitis contig 2.2 (accession number AAEC02000002). Nineteen isolates of Coccidioides spp. were examined for the developed primers (Table 1). The PCR system with the specific primer pairs was able to amplify the DNA fragment of the expected size from DNAs of C. immitis (Fig. 1). For specificity testing, 137 isolates of 52 fungal species were examined (Table 2). As a result of PCR using DNAs from these fungi, the primers were proved not to cross-amplify with major pathogenic fungi and related ones, such as Arthrographis kalrae, Chrysosporium spp., Geotrichum candidum, Malbranchea spp., Paracoccidioides brasiliensis, and Trichosporon asahii. The Coccidioides diagnostics based on a proline-rich antigen (2, 6) or internal transcribed spacer region (5, 7) have been reported so far. However, Bialek (1) has pointed out that the possibility of cross-amplification of human and murine DNA by the ITS primers (7) was not excluded. Since no amplification of human DNA by the primers in this report was detected (data not shown), we have developed a useful PCR system applicable for use in clinical diagnosis.
Surprisingly, two different mobilities of the DNA fragment were observed (Fig. 1) when the primers were tested for all Coccidioides spp. DNA available in Japan (Table 1). The DNA fragment amplified from C. posadasii was obviously shorter than that from C. immitis. Nucleotide sequence analysis of the amplified DNA revealed that the amplicon from C. posadasii had a contiguous deletion of 86 bp compared to that from C. immitis (Fig. 2). Therefore, such a large deletion contributed to the convenient distinction of two very close species, C. immitis and C. posadasii, which had previously been difficult to distinguish. In actual distinction on an agarose gel, standard amplicons of C. immitis and C. posadasii should be run as controls because of the close proximity of the two bands. Two methods for differentiating C. immitis and C. posadasii are currently being used: the lengths of the microsatellite loci and the single-nucleotide polymorphisms within several enzymes (3). Both methods require highly skilled molecular biological techniques at a relatively high cost. The PCR system with the primers we developed might facilitate operation and provide high-throughput handling. Thus, this will provide high value in epidemiology, such as tracing the route of laboratory-acquired infection or analyzing a pandemic that might occur in the future. Whether these primers can be clinically applied remains to be seen. Further development will contribute to the early diagnosis of coccidioidomycosis.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent technical assistance of Yuki Utena-Abe.
This study was supported by Health and Labour Sciences Research Grants for Research on Emerging and Re-emerging Infectious Diseases from Japan's Ministry of Health, Labour and Welfare. This study was also supported by the National Bioresource Project-Pathogenic Microorganisms of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
Bialek, R. 2005. Amplification of coccidioidal DNA in clinical specimens by PCR. J. Clin. Microbiol. 43:1492-1493.
Bialek, R., J. Kern, T. Herrmann, R. Tijerina, L. Cecenas, U. Reischl, and G. M. Gonzalez. 2004. PCR assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen. J. Clin. Microbiol. 42:778-783.
Fisher, M. C., G. L. Koenig, T. J. White, G. San-Blas, R. Negroni, I. G. Alvarez, B. Wanke, and J. W. Taylor. 2001. Biogeographic range expansion into South America by Coccidioides immitis mirrors New World patterns of human migration. Proc. Natl. Acad. Sci. USA 98:4558-4562.
Fisher, M. C., G. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73-84.
Greene, D. R., G. L. Koenig, M. C. Fisher, and J. W. Taylor. 2000. Soil isolation and molecular identification of Coccidioides immitis. Mycologia 92:406-410.
Johannesson, H., P. Vidal, J. Guarro, R. A. Herr, G. T. Cole, and J. W. Taylor. 2004. Positive directional selection in the proline-rich antigen (PRA) gene among the human pathogenic fungi Coccidioides immitis, C. posadasii and their closest relatives. Mol. Biol. Evol. 21:1134-1145.
Johnson, S. M., K. A. Simmons, and D. Pappagianis. 2004. Amplification of coccidioidal DNA in clinical specimens by PCR. J. Clin. Microbiol. 42:1982-1985.
Lindsley, M. D., S. F. Hurst, N. J. Iqbal, and C. J. Morrison. 2001. Rapid identification of dimorphic and yeast-like fungal pathogens using specific DNA probes. J. Clin. Microbiol. 39:3505-3511.
Sandhu, G. S., B. C. Kline, L. Stockman, and G. D. Roberts. 1995. Molecular probes for diagnosis of fungal infections. J. Clin. Microbiol. 33:2913-2919.
Sano, A., M. Miyaji, K. Kamei, Y. Mikami, and K. Nishimura. Reexamination of Coccidioides spp. reserved in the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, based on a multiple gene analysis. Nippon Ishinkin Gakkai Zasshi (Jpn. J. Med. Mycol.), in press.
Shubitz, L., T. Peng, R. Perrill, J. Simons, K. Orsborn, and J. N. Galgiani. 2002. Protection of mice against Coccidioides immitis intranasal infection by vaccination with recombinant antigen 2/PRA. Infect. Immun. 70:3287-3289.
Stockman, L., K. A. Clark, J. M. Hunt, and G. D. Roberts. 1993. Evaluation of commercially available acridinium ester-labeled chemiluminescent DNA probes for culture identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum. J. Clin. Microbiol. 31:845-850.(Takashi Umeyama, Ayako Sa)
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-8673, Japan
ABSTRACT
We developed a pair of primers that specifically identifies Coccidioides species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing Coccidioides immitis and Coccidioides posadasii by simply comparing the amplicon sizes on an agarose gel.
TEXT
Coccidioidomycosis, a fungal respiratory disease of humans caused by Coccidioides species, is endemic to arid areas of the Americas. Two species of Coccidioides are now recognized, whereas until recently, coccidioidomycosis was attributed to only one species, Coccidioides immitis (4). Coccidioides posadasii, formerly known as the non-California C. immitis strain, is found mainly in Texas, Arizona, and regions of endemicity outside of the United States, whereas C. immitis is found primarily in the Central Valley of California. These two species can be divided based on single-nucleotide polymorphisms and the size of microsatellites (3, 4), although the colony morphologies, growth rates, and clinical presentations are almost identical. Because identification of Coccidioides spp. carries with it a great deal of risk, molecular diagnosis without culturing has long been expected. Many researchers have explored nucleic acid detection for the diagnosis of coccidioidomycosis (2, 7-9, 11, 12).
For isolates of C. immitis and C. posadasii, listed in Table 1, the same DNA samples previously described by Sano et al. (10) were used. For non-Coccidioides fungal isolates, listed in Table 2, DNA isolation was performed using a DEXPAT DNA extraction kit (Takara Bio Inc., Japan). PCR was performed with approximately 10 ng of extracted DNA in a 20-μl reaction volume consisting of LA Taq buffer II (Mg2+ plus) (Takara Bio Inc.), 200 μM deoxynucleoside triphosphates, 2.5 U of ExTaq DNA polymerase (Takara Bio Inc.), and 10 pmol of each primer. One cycle at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 45 s with a final extension step at 72°C for 3 min was performed in a PTC-200 DNA Engine thermal cycler (Bio-Rad). The amplified DNA must be handled carefully in order to avoid amplicon contamination. The universal fungal primers ITS1 and ITS4 were used in all DNA samples to verify the efficiency of the test and to ensure that there was no PCR inhibition in the DNA samples (8). Ten microliters of each PCR product was electrophoresed through 2% agarose gel and visualized with a UV light after ethidium bromide or SYBR Safe (Invitrogen) staining. The PCR products were purified from gel with a NucleoSpin Extract II kit (Macherey-Nagel, Germany). Nucleotide sequences were determined using a BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) and an ABI 3130 genetic analyzer (Applied Biosystems).
Empirically, the construction of diagnostic primers is based on a nucleotide sequence encoding conserved enzymes or rRNA. However, we left the matter to chance: we repeated the primer construction based on randomly selected regions and verification by actual PCR experiments. In step 1, we obtained a text file containing the C. immitis genome sequence (http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis/). A nucleotide sequence corresponding to the 240- to 720-bp length was randomly selected from the genome database file. We designed 20-mer forward and reverse primers, which were expected to amplify the randomly selected region. In step 2, we examined whether these two primers could amplify DNA fragments of the anticipated size from Coccidioides spp. In step 3, we tested whether the primers that could amplify Coccidioides DNA in step 2 were unable to amplify Candida albicans and Aspergillus fumigatus DNA. By repeating these steps on 64 selected regions, we nominated one pair of primers for more detailed examination. Since this experimental primer design led to our successful product, mentioned below, this strategy should be a powerful method for the development of diagnostic primers.
The selected primers were Coi9-1F (5'-TACGGTGTAATCCCGATACA-3') and Coi9-1R (5'-GGTCTGAATGATCTGACGCA-3'). The selected primer set was constructed to amplify a 720-bp amplicon that corresponds to nucleotide position 660313 to 661032 of C. immitis contig 2.2 (accession number AAEC02000002). Nineteen isolates of Coccidioides spp. were examined for the developed primers (Table 1). The PCR system with the specific primer pairs was able to amplify the DNA fragment of the expected size from DNAs of C. immitis (Fig. 1). For specificity testing, 137 isolates of 52 fungal species were examined (Table 2). As a result of PCR using DNAs from these fungi, the primers were proved not to cross-amplify with major pathogenic fungi and related ones, such as Arthrographis kalrae, Chrysosporium spp., Geotrichum candidum, Malbranchea spp., Paracoccidioides brasiliensis, and Trichosporon asahii. The Coccidioides diagnostics based on a proline-rich antigen (2, 6) or internal transcribed spacer region (5, 7) have been reported so far. However, Bialek (1) has pointed out that the possibility of cross-amplification of human and murine DNA by the ITS primers (7) was not excluded. Since no amplification of human DNA by the primers in this report was detected (data not shown), we have developed a useful PCR system applicable for use in clinical diagnosis.
Surprisingly, two different mobilities of the DNA fragment were observed (Fig. 1) when the primers were tested for all Coccidioides spp. DNA available in Japan (Table 1). The DNA fragment amplified from C. posadasii was obviously shorter than that from C. immitis. Nucleotide sequence analysis of the amplified DNA revealed that the amplicon from C. posadasii had a contiguous deletion of 86 bp compared to that from C. immitis (Fig. 2). Therefore, such a large deletion contributed to the convenient distinction of two very close species, C. immitis and C. posadasii, which had previously been difficult to distinguish. In actual distinction on an agarose gel, standard amplicons of C. immitis and C. posadasii should be run as controls because of the close proximity of the two bands. Two methods for differentiating C. immitis and C. posadasii are currently being used: the lengths of the microsatellite loci and the single-nucleotide polymorphisms within several enzymes (3). Both methods require highly skilled molecular biological techniques at a relatively high cost. The PCR system with the primers we developed might facilitate operation and provide high-throughput handling. Thus, this will provide high value in epidemiology, such as tracing the route of laboratory-acquired infection or analyzing a pandemic that might occur in the future. Whether these primers can be clinically applied remains to be seen. Further development will contribute to the early diagnosis of coccidioidomycosis.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent technical assistance of Yuki Utena-Abe.
This study was supported by Health and Labour Sciences Research Grants for Research on Emerging and Re-emerging Infectious Diseases from Japan's Ministry of Health, Labour and Welfare. This study was also supported by the National Bioresource Project-Pathogenic Microorganisms of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
Bialek, R. 2005. Amplification of coccidioidal DNA in clinical specimens by PCR. J. Clin. Microbiol. 43:1492-1493.
Bialek, R., J. Kern, T. Herrmann, R. Tijerina, L. Cecenas, U. Reischl, and G. M. Gonzalez. 2004. PCR assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen. J. Clin. Microbiol. 42:778-783.
Fisher, M. C., G. L. Koenig, T. J. White, G. San-Blas, R. Negroni, I. G. Alvarez, B. Wanke, and J. W. Taylor. 2001. Biogeographic range expansion into South America by Coccidioides immitis mirrors New World patterns of human migration. Proc. Natl. Acad. Sci. USA 98:4558-4562.
Fisher, M. C., G. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73-84.
Greene, D. R., G. L. Koenig, M. C. Fisher, and J. W. Taylor. 2000. Soil isolation and molecular identification of Coccidioides immitis. Mycologia 92:406-410.
Johannesson, H., P. Vidal, J. Guarro, R. A. Herr, G. T. Cole, and J. W. Taylor. 2004. Positive directional selection in the proline-rich antigen (PRA) gene among the human pathogenic fungi Coccidioides immitis, C. posadasii and their closest relatives. Mol. Biol. Evol. 21:1134-1145.
Johnson, S. M., K. A. Simmons, and D. Pappagianis. 2004. Amplification of coccidioidal DNA in clinical specimens by PCR. J. Clin. Microbiol. 42:1982-1985.
Lindsley, M. D., S. F. Hurst, N. J. Iqbal, and C. J. Morrison. 2001. Rapid identification of dimorphic and yeast-like fungal pathogens using specific DNA probes. J. Clin. Microbiol. 39:3505-3511.
Sandhu, G. S., B. C. Kline, L. Stockman, and G. D. Roberts. 1995. Molecular probes for diagnosis of fungal infections. J. Clin. Microbiol. 33:2913-2919.
Sano, A., M. Miyaji, K. Kamei, Y. Mikami, and K. Nishimura. Reexamination of Coccidioides spp. reserved in the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, based on a multiple gene analysis. Nippon Ishinkin Gakkai Zasshi (Jpn. J. Med. Mycol.), in press.
Shubitz, L., T. Peng, R. Perrill, J. Simons, K. Orsborn, and J. N. Galgiani. 2002. Protection of mice against Coccidioides immitis intranasal infection by vaccination with recombinant antigen 2/PRA. Infect. Immun. 70:3287-3289.
Stockman, L., K. A. Clark, J. M. Hunt, and G. D. Roberts. 1993. Evaluation of commercially available acridinium ester-labeled chemiluminescent DNA probes for culture identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum. J. Clin. Microbiol. 31:845-850.(Takashi Umeyama, Ayako Sa)