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Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction
MICROBIOLOGY LETTERS
ELSEVIER
FEMS Microbiology Letters 145 (1996) 23-26
Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction D. Liu *, S. Coloe, Melhaurne Pathology,
S. Lloyd
Jones, R. Baird, J. Pedersen
32 Smith Street, Collingwood, Vie. 3066, Australia
Received 28 May 1996: accepted 10 June 1996
Abstract Can&da species are opportunistic human pathogens capable of causing a variety of clinical diseases. Rapid and precise identification of Cundidu to species level is essential for effective treatment and management strategies. Conventional diagnosis of candidiasis is sometimes slow and variable. By using a random primer 5’-ACGGGCCAGT-3’ in arbitrarily primed polymerase chain reaction, seven common Candida species (i.e., C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. lipolytica. C. tropicalis and C. (Torulopsis) glabrata) produced characteristic DNA band patterns, which enabled rapid determination to species level among them. Further analysis of the nucleotide sequences of Cundidu specific fragments should improve our understanding of the molecular structures and possible functions of the gene regions involved. Ke_v~ords: Cundida: Yeast; Fungus; .4rbitrarily primed polymerase chain reaction: Speciation
1. Introduction The anamorphic yeasts in the genus Candida are opportunistic human pathogens, and several Candida species are present on the mucosal surfaces such as the mouth, gastrointestinal tract and vagina as human commensals. Depending on the health status of individuals, Candida species are capable of producing a diversity of clinical diseases, ranging from superficial to invasive infections. While systematic Candida infections frequently occur in immunosuppressed or burn patients, mucosal candidiasis is also a frequent cause of morbidity. The current laboratory diagnosis of candidosis is * Corresponding author. Tel: +61 (3) 9287 7746; Fax: +61 (3) 9287 7710.
based on examination by microscopy and culture. Under normal circumstances, identification of candidemia by these methods can take 2-5 days. However, because of the slow growth of some Candida isolates from clinical specimens, species identification can take longer. Furthermore, as common identification procedures measure the phenotypic characteristics of Cundida species, the results can be variable and sometimes ambiguous. The pathogenicity and antifungal susceptibility of Candidu species may vary from one to another, which further necessitates a rapid and specific identification to species level in order to make appropriate management decisions. Detection and species identification of Candida isolates by polymerase chain reaction (PCR) based assays possess several advantages over the conventional microscopy and culture procedures. The PCR
037%1097/96/$12.00 Copyright 0 1996 Published by Elsevier Science B.V. All rights reserved PUSO378-1097(96)00376-X
24
D Liu ct al. I FEMS Microbiology
assays are rapid, and can generally be completed in a day. Moreover, they are precise, as they detect differences at the genetic level. In the past few years, a number of PCR based procedures have been reported for improved identification of Candida species [14]. Nevertheless, most of these PCR procedures require additional manipulations such as restriction enzyme digestion or DNA sequencing analysis after amplification, which can often be cumbersome and time-consuming, preventing widespread adoption in clinical laboratories. The application of arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) technology [5-71 has made the genetic differentiation of Candida species a simpler task [8,9]. In addition to obviating the need to know the detailed sequences of the gene regions concerned, the AP-PCR or RAPD does not involve further steps after amplification [557]. Using the RAPD technique. Lehmann et al. [8] demonstrated that DNA products generated from common Candida species such as C. parapsilosis, C. tropicalis, C. glabrata and C. lusitaniae formed distinct band patterns upon gel electrophoresis, therefore permitting their rapid differentiation. In this study, we report the use of a different random primer, 5’-ACGGGCCAGT-3’, in arbitrarily primed PCR for genetic speciation of C. alhicans, C. guilliermondii, C. parupsilosis, C. krusei, C. lipolytica, C. tropicalis and C. (Torulopsis) glubrata. The amplified DNA products from these Cundidu species showed characteristic band patterns, which were somewhat different from those reported [8,9], suggesting possible involvement of other gene regions. Further analysis of the nucleotide sequences of Candida specific fragments produced in this study may help reveal the molecular structures and possible functions of the gene regions involved.
2. Materials and methods 2.1. Organisms Candida species from clinical samples were cultured onto Sabourand agar (Oxoid) and identified to species level using VITEK Yeast Biochemical Card (Vitek Systems, Missouri, USA) and API20 C AUX Yeast Identification System (BioMCrieux,
Letters 145 (1996)
23-26
Lyon, France) in accordance with manufacturers’ instructions. A number of other fungi such as dermatophytes were obtained as described elsewhere [lo] and used for comparison. In total, 24 Candidu and other fungi were examined in the present study. These comprised four Candida albicans, three C. guilliermondii, three C. parapsilosis, two C. krusei, two C. lipolytica, two C. tropicalis, two C. (Torulopsis) glabrata, one Trichophyton rubrum, one T. mentagrophytes, one T. tonswans, one Epidermophyton jloccosum and one Microsporum canis isolates. 2.2. DNA extraction Genomic DNA from Candida species was prepared using a rapid method reported earlier [lo]. Namely, yeasts (from a full 9 cm Sabourand agar plate) were collected into a 15 ml tube with 4 ml of SET buffer (75 mM NaCl. 2.5 mM EDTA, 20 mM Tris-HCl pH 8). The yeast cells were disrupted by mechanical shearing through an 18 gauge needle several times. Following addition of 1 ml of 2.5 mg/ ml lysozyme (freshly made), the tubes were incubated at room temperature for 1 h. 25 ul of 10 mg/ml RNase was added, and the tubes incubated at 37°C for 30 min. Next 500 ul of 10% SDS and 50 ul of 10 mg/ml proteinase K were added and the tubes were incubated at 50°C for 1 h. Then 2 ml of 6 M NaCl and 7.5 ml chloroform were added, mixed gently in a rotating shaker for 10 min and then centrifuged at 3500 rpm for 20 min. The supernatant was transferred to a new 15 ml tube, and the DNA was precipitated with 7.5 ml isopropanol by gentle inversion and subsequent centrifugation at 3500 rpm for 20 min at 4°C. After washing with 70% ethanol, the DNA was vacuum-dried and dissolved in 1 ml of 1 X TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0). The DNA concentration was determined by absorption at UV 260 nm in a spectrophotometer, and an aliquot of the purified DNA was diluted to about 10 nglyl in sterile water which was stored at -20°C until used. Genomic DNA from other fungi was also prepared similarly. 2.3. Arbitrarily primed PCR The arbitrarily primed PCR was carried out in a total volume of 50 ul reaction mixture. The reaction
D Liar et a/. / FEMS Microbiology Lettrrs 145 (1996~ 23-26
25
obcdefghijklmnopqrstuvwx
Fig. 1. Examination of the arbitrarily primed PCR products amplified from Candida and other fungi by agarose gel electrophoresis. Lanes ad contained the amplified DiiA products from C. alhicans isolates; lanes e-g, C. guilliermondii: lanes h-j, C. p~lrqsilosrs: lanes k, I, C. krusei; lanes m, n. C. lipolytim; lanes o. p. C. tropicalir; lanes q, r. C. ~Torulopsis) gkuhrata; lane s, Trichophyton rubrum; lane t, T mentagrophytes; lane u, T. ton.surans; lane x’, Epidwmophwon ~%~cosum; lane w, Microsporum canis; and lane x. negative control with no template DNA. The numbers on the left are molecular weight markers in kb (h HindIII+EcoRI).
mixture consisted of approximately 10 ng genomic DNA, 50 mM KCl, 10 mM Tris-HCI (pH 9.0), 10 mM NaCl, 0.5 mM DTT. 2.5 mM MgC12, 0.1% Triton X-100, 200 ,uM of each dNTP (dATP, dCTP, dGTP and dTTP), 1 U of Taq DNA polymerase (Life Technologies) and 20 pmol of one of 50 different random decamers (Operon Technologies, USA). The reaction mixture was incubated in a GeneAmpm PCR System 9600 (Perkin Elmer, Roche, USA) using the following protocol: 3 cycles of 94°C for 60 s, 36°C for 45 s, 12°C for 90 s and 32 cycles of 94°C for 30 s, 36°C for 45 s, 72°C for 90 s. A tube with no template DNA was included as a negative control. Upon completion, 5 ul of 10 X DNA sample buffer containing bromophenol and glycerol was added to each tube, from which approximately 10 ul was electrophoresed (at 5 V/cm) in a 1.5% agarose gel in I X TAE buffer in the presence of ethidium bromide, and subsequently examined under UV light.
3. Results and discussion Of the 50 random decamers used in the AP-PCR, one primer, 5’-ACGGGCCAGT-3’, amplified bands of 1.2 and 0.7 kb in DNA from four Cundidu albicans isolates (Fig. la-d) as well as an addition band of 0.8 kb in one of the four isolates (Fig. lb). This primer also generated bands of 1.4, 1.0: 0.8 and 0.6 kb in C, guilliermondii DNA (Fig. le-g): bands of 3.0, 1.l and 0.6 kb in C. parapsilosis DNA (Fig. lhj); bands of 3.0, 2.0, 1.2, 0.9, 0.7 and 0.4 kb in C. krusei DNA (Fig. lk,l): bands of 2.1, 1.3 and 1.2 kb in C. lipolytica DNA (Fig. lm,n); bands of 1.4, 1.l
and 0.6 kb in C. tropicalis DNA (Fig. 10,~); bands of 1.6 and 0.8 kb in C. (Torulopsisj glabrata DNA (Fig. lq,r): bands of 3.0, 1.6, 1.4, 0.6 and 0.4 kb in Trichophyton rubrum DNA (Fig. 1s); bands of 3.0, 1.2 and 0.8 kb in T. mentugrophytes DNA (Fig. It); bands of 1.5 and 0.8 kb in T. tonswans DNA (Fig. lu); bands of 1.4 and 0.9 kb in Epidermophyton jlo~~o~~m DNA (Fig. Iv); bands of 2.4, 0.9 and 0.5 kb in Microsporum cunis DNA (Fig. Iw) and no band in the negative (no template DNA) control (Fig. lx). The observations that DNA products amplified from various Candida species using the random primer 5’-ACGGGCCAGT-3’ in the arbitrarily primed PCR displayed characteristic band patterns may provide a useful criterion for rapid speciation of Candida species. The ability to detect and identify rapidly and precisely various common Candida species through the arbitrarily primed PCR has further simplified the genetic speciation of yeasts [8,9]. The AP-PCR is reasonably robust, and identical results have been produced over repeat runs on different days (data not shown). It shall be of interest to see if other Candida species than the seven examined can also be distinguished using the random primer 5’ACGGGCCAGT-3’ in the AP-PCR. In addition, the extension of this AP-PCR to the detection and differentiation of various Candida species directly from clinical materials such as blood will further speed up the identification process. The DNA fragments amplified from the seven Cundida species using the random primer 5’ACGGGCCAGT-3’ appeared to be different from those described previously [8,9] in terms of sizes and band patterns. It is possible that the random
26
D. Liu et al. I FEMS
Microbiology
primer 5’-ACGGGCCAGT-3’ may recognize and amplify some gene regions in Candida species distinct from those by other primers [8,9]. Nevertheless, it is clear from the present study that the gene region(s) in the Candida genome recognized by the random primer 5’-ACGGGCCATG are probably evolutionarily variable as the sizes of the amplified DNA products differ from species to species. The elucidation of the nucleotide base sequences of the Candida specific DNA fragments may shed light on the molecular structures and possible function of the region(s) involved, and lead to improved understanding of the genetic relationship among various Candida species as well as other related fungi.
References [l] Jordan, J.A. (1994) PCR identification of four medically important Candidu species by using a single primer pair. J. Clin. Microbial. 32, 296222967. [2] Burgener-Kairnz, P., Zuber, J.P., Jaunin, P., Buchman, T.G., Bille, J. and Rossier, M. (1994) Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-a-demethylase (LlAl) gene fragment. J. Clin. Microbial. 32, 1902-1907.
Letters
I45 (1996)
23-26
[3] Williams, D.W., Wilson, M.J., Lewis, M.A.O. and Potts, J.C. (1995) Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J. Clin. Microbial. 33, 24762479. [4] Thanos, M., Schoian, G., Meyer, W., Schweynoch. C., Graser, Y., Mitchell, T.G., Presber, W. and Tietz, H.J. (1996) Rapid identification of Candida species by DNA fingerprinting with PCR. J. Clin. Microbial. 34, 615-621. [S] Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213-7218. [6] Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531-6535. [7] Caetano-Anolles, G., Bassam, B.J. and Gresshoff, P.M. (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. BiolTechnology 9, 553-557. [S] Lehmann, P.F., Lin, D. and Laker, B.A. (1992) Genotype identification and characterisation of species and strains with genus Candida by using random amplified polymorphic DNA. J. Clin. Microbial. 30, 3249-3254. [9] Lin, D. and Lehmann, P.F. (1995) Random amplified polymorphic DNA for strain delineation within Cundida tropic&. J. Med. Vet. Mycol. 33, 241-246. [IO] Liu, D., Coloe, S., Pedersen. J. and Baird. R. (1996) Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyron dermatophytes. FEMS Microbial. Lett. 136, 1477150.
ELSEVIER
FEMS Microbiology Letters 145 (1996) 23-26
Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction D. Liu *, S. Coloe, Melhaurne Pathology,
S. Lloyd
Jones, R. Baird, J. Pedersen
32 Smith Street, Collingwood, Vie. 3066, Australia
Received 28 May 1996: accepted 10 June 1996
Abstract Can&da species are opportunistic human pathogens capable of causing a variety of clinical diseases. Rapid and precise identification of Cundidu to species level is essential for effective treatment and management strategies. Conventional diagnosis of candidiasis is sometimes slow and variable. By using a random primer 5’-ACGGGCCAGT-3’ in arbitrarily primed polymerase chain reaction, seven common Candida species (i.e., C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. lipolytica. C. tropicalis and C. (Torulopsis) glabrata) produced characteristic DNA band patterns, which enabled rapid determination to species level among them. Further analysis of the nucleotide sequences of Cundidu specific fragments should improve our understanding of the molecular structures and possible functions of the gene regions involved. Ke_v~ords: Cundida: Yeast; Fungus; .4rbitrarily primed polymerase chain reaction: Speciation
1. Introduction The anamorphic yeasts in the genus Candida are opportunistic human pathogens, and several Candida species are present on the mucosal surfaces such as the mouth, gastrointestinal tract and vagina as human commensals. Depending on the health status of individuals, Candida species are capable of producing a diversity of clinical diseases, ranging from superficial to invasive infections. While systematic Candida infections frequently occur in immunosuppressed or burn patients, mucosal candidiasis is also a frequent cause of morbidity. The current laboratory diagnosis of candidosis is * Corresponding author. Tel: +61 (3) 9287 7746; Fax: +61 (3) 9287 7710.
based on examination by microscopy and culture. Under normal circumstances, identification of candidemia by these methods can take 2-5 days. However, because of the slow growth of some Candida isolates from clinical specimens, species identification can take longer. Furthermore, as common identification procedures measure the phenotypic characteristics of Cundida species, the results can be variable and sometimes ambiguous. The pathogenicity and antifungal susceptibility of Candidu species may vary from one to another, which further necessitates a rapid and specific identification to species level in order to make appropriate management decisions. Detection and species identification of Candida isolates by polymerase chain reaction (PCR) based assays possess several advantages over the conventional microscopy and culture procedures. The PCR
037%1097/96/$12.00 Copyright 0 1996 Published by Elsevier Science B.V. All rights reserved PUSO378-1097(96)00376-X
24
D Liu ct al. I FEMS Microbiology
assays are rapid, and can generally be completed in a day. Moreover, they are precise, as they detect differences at the genetic level. In the past few years, a number of PCR based procedures have been reported for improved identification of Candida species [14]. Nevertheless, most of these PCR procedures require additional manipulations such as restriction enzyme digestion or DNA sequencing analysis after amplification, which can often be cumbersome and time-consuming, preventing widespread adoption in clinical laboratories. The application of arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) technology [5-71 has made the genetic differentiation of Candida species a simpler task [8,9]. In addition to obviating the need to know the detailed sequences of the gene regions concerned, the AP-PCR or RAPD does not involve further steps after amplification [557]. Using the RAPD technique. Lehmann et al. [8] demonstrated that DNA products generated from common Candida species such as C. parapsilosis, C. tropicalis, C. glabrata and C. lusitaniae formed distinct band patterns upon gel electrophoresis, therefore permitting their rapid differentiation. In this study, we report the use of a different random primer, 5’-ACGGGCCAGT-3’, in arbitrarily primed PCR for genetic speciation of C. alhicans, C. guilliermondii, C. parupsilosis, C. krusei, C. lipolytica, C. tropicalis and C. (Torulopsis) glubrata. The amplified DNA products from these Cundidu species showed characteristic band patterns, which were somewhat different from those reported [8,9], suggesting possible involvement of other gene regions. Further analysis of the nucleotide sequences of Candida specific fragments produced in this study may help reveal the molecular structures and possible functions of the gene regions involved.
2. Materials and methods 2.1. Organisms Candida species from clinical samples were cultured onto Sabourand agar (Oxoid) and identified to species level using VITEK Yeast Biochemical Card (Vitek Systems, Missouri, USA) and API20 C AUX Yeast Identification System (BioMCrieux,
Letters 145 (1996)
23-26
Lyon, France) in accordance with manufacturers’ instructions. A number of other fungi such as dermatophytes were obtained as described elsewhere [lo] and used for comparison. In total, 24 Candidu and other fungi were examined in the present study. These comprised four Candida albicans, three C. guilliermondii, three C. parapsilosis, two C. krusei, two C. lipolytica, two C. tropicalis, two C. (Torulopsis) glabrata, one Trichophyton rubrum, one T. mentagrophytes, one T. tonswans, one Epidermophyton jloccosum and one Microsporum canis isolates. 2.2. DNA extraction Genomic DNA from Candida species was prepared using a rapid method reported earlier [lo]. Namely, yeasts (from a full 9 cm Sabourand agar plate) were collected into a 15 ml tube with 4 ml of SET buffer (75 mM NaCl. 2.5 mM EDTA, 20 mM Tris-HCl pH 8). The yeast cells were disrupted by mechanical shearing through an 18 gauge needle several times. Following addition of 1 ml of 2.5 mg/ ml lysozyme (freshly made), the tubes were incubated at room temperature for 1 h. 25 ul of 10 mg/ml RNase was added, and the tubes incubated at 37°C for 30 min. Next 500 ul of 10% SDS and 50 ul of 10 mg/ml proteinase K were added and the tubes were incubated at 50°C for 1 h. Then 2 ml of 6 M NaCl and 7.5 ml chloroform were added, mixed gently in a rotating shaker for 10 min and then centrifuged at 3500 rpm for 20 min. The supernatant was transferred to a new 15 ml tube, and the DNA was precipitated with 7.5 ml isopropanol by gentle inversion and subsequent centrifugation at 3500 rpm for 20 min at 4°C. After washing with 70% ethanol, the DNA was vacuum-dried and dissolved in 1 ml of 1 X TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0). The DNA concentration was determined by absorption at UV 260 nm in a spectrophotometer, and an aliquot of the purified DNA was diluted to about 10 nglyl in sterile water which was stored at -20°C until used. Genomic DNA from other fungi was also prepared similarly. 2.3. Arbitrarily primed PCR The arbitrarily primed PCR was carried out in a total volume of 50 ul reaction mixture. The reaction
D Liar et a/. / FEMS Microbiology Lettrrs 145 (1996~ 23-26
25
obcdefghijklmnopqrstuvwx
Fig. 1. Examination of the arbitrarily primed PCR products amplified from Candida and other fungi by agarose gel electrophoresis. Lanes ad contained the amplified DiiA products from C. alhicans isolates; lanes e-g, C. guilliermondii: lanes h-j, C. p~lrqsilosrs: lanes k, I, C. krusei; lanes m, n. C. lipolytim; lanes o. p. C. tropicalir; lanes q, r. C. ~Torulopsis) gkuhrata; lane s, Trichophyton rubrum; lane t, T mentagrophytes; lane u, T. ton.surans; lane x’, Epidwmophwon ~%~cosum; lane w, Microsporum canis; and lane x. negative control with no template DNA. The numbers on the left are molecular weight markers in kb (h HindIII+EcoRI).
mixture consisted of approximately 10 ng genomic DNA, 50 mM KCl, 10 mM Tris-HCI (pH 9.0), 10 mM NaCl, 0.5 mM DTT. 2.5 mM MgC12, 0.1% Triton X-100, 200 ,uM of each dNTP (dATP, dCTP, dGTP and dTTP), 1 U of Taq DNA polymerase (Life Technologies) and 20 pmol of one of 50 different random decamers (Operon Technologies, USA). The reaction mixture was incubated in a GeneAmpm PCR System 9600 (Perkin Elmer, Roche, USA) using the following protocol: 3 cycles of 94°C for 60 s, 36°C for 45 s, 12°C for 90 s and 32 cycles of 94°C for 30 s, 36°C for 45 s, 72°C for 90 s. A tube with no template DNA was included as a negative control. Upon completion, 5 ul of 10 X DNA sample buffer containing bromophenol and glycerol was added to each tube, from which approximately 10 ul was electrophoresed (at 5 V/cm) in a 1.5% agarose gel in I X TAE buffer in the presence of ethidium bromide, and subsequently examined under UV light.
3. Results and discussion Of the 50 random decamers used in the AP-PCR, one primer, 5’-ACGGGCCAGT-3’, amplified bands of 1.2 and 0.7 kb in DNA from four Cundidu albicans isolates (Fig. la-d) as well as an addition band of 0.8 kb in one of the four isolates (Fig. lb). This primer also generated bands of 1.4, 1.0: 0.8 and 0.6 kb in C, guilliermondii DNA (Fig. le-g): bands of 3.0, 1.l and 0.6 kb in C. parapsilosis DNA (Fig. lhj); bands of 3.0, 2.0, 1.2, 0.9, 0.7 and 0.4 kb in C. krusei DNA (Fig. lk,l): bands of 2.1, 1.3 and 1.2 kb in C. lipolytica DNA (Fig. lm,n); bands of 1.4, 1.l
and 0.6 kb in C. tropicalis DNA (Fig. 10,~); bands of 1.6 and 0.8 kb in C. (Torulopsisj glabrata DNA (Fig. lq,r): bands of 3.0, 1.6, 1.4, 0.6 and 0.4 kb in Trichophyton rubrum DNA (Fig. 1s); bands of 3.0, 1.2 and 0.8 kb in T. mentugrophytes DNA (Fig. It); bands of 1.5 and 0.8 kb in T. tonswans DNA (Fig. lu); bands of 1.4 and 0.9 kb in Epidermophyton jlo~~o~~m DNA (Fig. Iv); bands of 2.4, 0.9 and 0.5 kb in Microsporum cunis DNA (Fig. Iw) and no band in the negative (no template DNA) control (Fig. lx). The observations that DNA products amplified from various Candida species using the random primer 5’-ACGGGCCAGT-3’ in the arbitrarily primed PCR displayed characteristic band patterns may provide a useful criterion for rapid speciation of Candida species. The ability to detect and identify rapidly and precisely various common Candida species through the arbitrarily primed PCR has further simplified the genetic speciation of yeasts [8,9]. The AP-PCR is reasonably robust, and identical results have been produced over repeat runs on different days (data not shown). It shall be of interest to see if other Candida species than the seven examined can also be distinguished using the random primer 5’ACGGGCCAGT-3’ in the AP-PCR. In addition, the extension of this AP-PCR to the detection and differentiation of various Candida species directly from clinical materials such as blood will further speed up the identification process. The DNA fragments amplified from the seven Cundida species using the random primer 5’ACGGGCCAGT-3’ appeared to be different from those described previously [8,9] in terms of sizes and band patterns. It is possible that the random
26
D. Liu et al. I FEMS
Microbiology
primer 5’-ACGGGCCAGT-3’ may recognize and amplify some gene regions in Candida species distinct from those by other primers [8,9]. Nevertheless, it is clear from the present study that the gene region(s) in the Candida genome recognized by the random primer 5’-ACGGGCCATG are probably evolutionarily variable as the sizes of the amplified DNA products differ from species to species. The elucidation of the nucleotide base sequences of the Candida specific DNA fragments may shed light on the molecular structures and possible function of the region(s) involved, and lead to improved understanding of the genetic relationship among various Candida species as well as other related fungi.
References [l] Jordan, J.A. (1994) PCR identification of four medically important Candidu species by using a single primer pair. J. Clin. Microbial. 32, 296222967. [2] Burgener-Kairnz, P., Zuber, J.P., Jaunin, P., Buchman, T.G., Bille, J. and Rossier, M. (1994) Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-a-demethylase (LlAl) gene fragment. J. Clin. Microbial. 32, 1902-1907.
Letters
I45 (1996)
23-26
[3] Williams, D.W., Wilson, M.J., Lewis, M.A.O. and Potts, J.C. (1995) Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J. Clin. Microbial. 33, 24762479. [4] Thanos, M., Schoian, G., Meyer, W., Schweynoch. C., Graser, Y., Mitchell, T.G., Presber, W. and Tietz, H.J. (1996) Rapid identification of Candida species by DNA fingerprinting with PCR. J. Clin. Microbial. 34, 615-621. [S] Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213-7218. [6] Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531-6535. [7] Caetano-Anolles, G., Bassam, B.J. and Gresshoff, P.M. (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. BiolTechnology 9, 553-557. [S] Lehmann, P.F., Lin, D. and Laker, B.A. (1992) Genotype identification and characterisation of species and strains with genus Candida by using random amplified polymorphic DNA. J. Clin. Microbial. 30, 3249-3254. [9] Lin, D. and Lehmann, P.F. (1995) Random amplified polymorphic DNA for strain delineation within Cundida tropic&. J. Med. Vet. Mycol. 33, 241-246. [IO] Liu, D., Coloe, S., Pedersen. J. and Baird. R. (1996) Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyron dermatophytes. FEMS Microbial. Lett. 136, 1477150.