Novel Approach to Designing Primers for Identification and Distinction of the Human Pathogenic Fungi Coccidioides immitis and Coccidioides posadasii by PCR Amplification

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.

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).

TABLE 1.

Coccidioides isolates used in this study

Lane no. in Fig. 1	Species	IFM no.a	Origin
1	C. posadasii	4935	Japan
2	C. posadasii	4945	Japan
3	C. posadasii	45809	United States
4	C. posadasii	45810	United States
5	C. posadasii	45811	United States
6	C. posadasii	45812	United States
7	C. posadasii	45813	United States
8	C. immitis	45815	United States
9	C. immitis	45816	United States
10	C. posadasii	45817	United States
11	C. immitis	46868	Japan
12	C. immitis	50992	United States
13	C. posadasii	50993	Japan
14	C. posadasii	50994	Japan
15	C. immitis	50995	Japan
16	C. posadasii	51112	Japan
17	C. posadasii	54194	Japan
18	C. posadasii	54195	Japan
19	C. posadasii	54196	Japan

^aIFM, Institute for Food Microbiology, Chiba University, Chiba, Japan.

TABLE 2.

Fungal species used as negative controls in this study

Species	No. of tested isolates
Absidia sp.	1
Alternaria sp.	1
Apinisia spp.	2
Arthroderma spp.	8
Arthrographis spp.	4
Aspergillus spp.	5
Auxarthron sp.	1
Basidiobolus sp.	1
Blastomyces sp.	1
Candida spp.	8
Chrysosporium spp.	4
Cladophialophora spp.	2
Cokeromyces sp.	1
Conidiobolus sp.	1
Cryptococcus spp.	5
Cunninghamella sp.	1
Emmonsia spp.	3
Epidermophyton sp.	1
Exophiala spp.	4
Fonsecaea sp.	1
Fusarium spp.	2
Geotrichum sp.	1
Gymnoascoideus spp.	3
Gymnoascus spp.	5
Histoplasma spp.	6
Hortaea sp.	1
Malassezia spp.	2
Malbranchea spp.	17
Microsporum spp.	2
Mortierella sp.	1
Mucor spp.	2
Neosartorya sp.	1
Paecilomyces spp.	2
Paracoccidioides spp.	6
Penicillium spp.	7
Phanerochaete spp.	2
Phialophora spp.	2
Prototheca sp.	1
Pseudallescheria sp.	1
Rhinocladiella sp.	1
Rhizomucor sp.	1
Rhizopus spp.	3
Scedosporium sp.	1
Schizophyllum sp.	2
Scopulariopsis sp.	1
Sporothrix sp.	1
Syncephalastrum sp.	1
Trichophyton spp.	3
Trichosporon sp.	1
Uncinocarpus sp.	1
Veronaea sp.	1
Zygorhynchus sp.	1

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.

FIG. 1.

PCR amplification of coccidioidal DNAs. Lanes M, DNA molecular weight marker used to estimate product size; lane W, distilled water used as a negative control; lanes 1 to 7, 10, 13, 14, and 16 to 19, C. posadasii; lanes 8, 9, 11, 12, and 15, C. immitis. The exact description of Coccidioides spp. is in Table 1.

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.

FIG. 2.

Nucleotide sequence alignment of DNA fragments amplified with Coi9-1F and Coi9-1R primers between C. posadasii IFM45809 (Cp45809) and IFM45810 (Cp45810) and C. immitis IFM45815 (Ci45815) and IFM45816 (Ci45816).