Biomedical subjects

Toshio Kanbe

Publications and source records attributed to Toshio Kanbe.

At least 19 recordsLinked to original sources

PubMed2006

Further investigations of glucose-6-phosphate dehydrogenase variants in Flores Island, eastern Indonesia.

We conducted field surveys for malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the eastern part of Flores Island, East Nusa Tenggara Province, Indonesia. A total of 1,108 volunteers (642 males and 466 females) belonging to three ethnic groups (Sikka, Ende and Bajo) were examined, and 55 G6PD-deficient individuals (38 males and 17 females) were detected. Among them, 50 samples were analyzed molecularly, in addition to three deficient cases in a Bajo family. In the Sikka population, G6PD Kaiping (1388G>A), one of the two common variants in the Chinese population, was unexpectedly found as the most dominant variant (11/22, 50.0%), followed by G6PD Chatham (1003G>A, 36.4%), G6PD Coimbra (592C>T, 9.1%) and G6PD Vanua Lava (383T>C, 4.5%). Frequency of G6PD Kaiping in the Sikka might be the highest among non-Chinese populations reported so far. In the Ende population, G6PD Vanua Lava (9/14, 64.3%) was the highest, followed by G6PD Kaiping (14.3%), G6PD Chinese-5 (1024C>T, 14.3%) and G6PD Chatham (7.1%). In the Bajo population, a total of 18 deficient cases were analyzed, and a novel mutation (844G>T) in exon 8 with a predicted amino acid change of 282 Asp>Tyr was found in a 7-year-old boy at a Bajo village near Maumere. This new Class II (mild type) variant was also confirmed in his mother and sister, and designated as G6PD Bajo Maumere. The missense mutation at the same nucleotide 844 has been known as G6PD Seattle/Lodi/Modena/Ferrara II, but this mutation is caused by a G>C substitution (282 Asp>His). In the Bajo population, G6PD Viangchan (871G>A, IVS 11 nt93 T>C, 1311C>T), the most common variant in continental Southeast Asian populations, was found to be the dominant (11/18, 61.1%), followed by G6PD Vanua Lava and the new variant (each 16.7%), and G6PD Coimbra (5.6%). These results strongly suggest that the Bajo peoples may have different ancestors from those for Sikka and Ende, and may be much closer to continental Southeast Asian populations. It is interesting that G6PD Canton (1376G>T), another common variant in Chinese, was not seen in the Flores population.

DNA Mutational Analysis↗

PubMed2006

Genotype analysis of Candida albicans isolates obtained from different body locations of patients with superficial candidiasis using PCRs targeting 25S rDNA and ALT repeat sequences of the RPS.

BACKGROUND: Several molecular biology-based genotyping techniques have been adapted for studying the molecular characteristics of Candida albicans strains, which constitute the majority of the etiologic agents in candidiasis. Recently, we reported a PCR system targeting 25S rDNA and ALT repeat sequences in the repetitive sequence (RPS) for genotyping of C. albicans. OBJECTIVE: To assess the potential of 25S rDNA and RPS-based genotyping for studying the molecular epidemiology of C. albicans, and define the genotypic relationship of C. albicans between invasive and non-invasive lesions in the same individual. METHODS: C. albicans strains were isolated from infected lesions and commensal sites, such as oral mucosa and/or feces, of patients with superficial candidiasis. The genomic DNAs were amplified by PCRs using P-I and P-II to determine the 25S rDNA- and RPS-based genotypes of the isolates. RESULTS: Genotype A:3 C. albicans constituted the majority of the isolates, followed by A:3/4 and B:3 C. albicans. There was usually one genotype of C. albicans per person. The genotypes of infected lesion isolates and non-infected oral mucosa and/or feces isolates were identical in the same individual, even in serially isolated C. albicans. CONCLUSION: The results indicate that our combined PCR technique using P-I and P-II is a potential tool for molecular typing of C. albicans, and reveal that the genotypes of isolates are identical in the same individual, independent of the infective and non-infective phases or the body location.

Adolescent↗

PubMed2006

Sequence variation in the T-cell epitopes of the Plasmodium falciparum circumsporozoite protein among field isolates is temporally stable: a 5-year longitudinal study in southern Vietnam.

In an effort to decipher the nature and extent of antigen polymorphisms of malaria parasites in a setting where malaria is hypomesoendemic, we conducted a 5-year longitudinal study (1998 to 2003) by sequencing the Th2R and Th3R epitopes of the circumsporozoite protein (CSP) of 142 Plasmodium falciparum field isolates from Bao Loc, Vietnam. Samples were collected during the high-transmission season, September through December 1998 (n = 43), as well as from July 2000 to August 2001 (n = 34), September 2001 to July 2002 (n = 33), and August 2002 to July 2003 (n = 32). Marked sequence diversity was noted during the high-transmission season in 1998, but no significant variation in allele frequencies was observed over the years (chi(2) = 70.003, degrees of freedom = 57, P = 0.116). The apparent temporal stability in allele frequency observed in this Bao Loc malaria setting may suggest that polymorphism in the Th2R and Th3R epitopes is not maintained by frequency-dependent immune selection. By including 36 isolates from Flores Island, Indonesia, and 19 isolates from Thaton, Myanmar, we investigated geographical patterns of sequence polymorphism for these epitopes in Southeast Asia; among the characterized isolates, a globally distributed variant appears to be predominant in Vietnam (75 of 142 isolates, or 52.8%) as well as in Myanmar (15 of 19 isolates, or 78.9%) and Indonesia (31 of 36 isolates, or 86.1%). Further analyses involving worldwide CSP sequences revealed distinct regional patterns, a finding which, together with the unique mutations observed here, may suggest a possible role for host or local factors in the generation of sequence diversity in the T-cell epitopes of CSP.

Animals↗

PubMed2005

Genotyping of Candida albicans on the basis of polymorphisms of ALT repeats in the repetitive sequence (RPS).

BACKGROUND: Candida albicans is one of the most important etiologic agents causing superficial and deep fungal infections. For prevention of candidiasis, it is important to develop a rapid system that discriminates C. albicans at the strain level. OBJECTIVE: To develop a system that can identify C. albicans at the strain level. METHODS: Genomic DNAs were purified from 179 clinical isolates of C. albicans, and were used as templates for PCR amplification of 25S rDNA and ALT repeats in repetitive sequences (RPSs). PCR products generated from ALT repeats were digested with EcoRI and/or ClaI in order to study the relationships between restriction profiles and amplification profiles. RESULTS: One hundred and seventy nine clinical isolates were grouped into genotypes A (92 isolates), B (38 isolates) and C (49 isolates) on the basis of their 25S rDNA, and each was further classified into five types (types 3, 4, 3/4, 2/3/4 and 3/4/5) by PCR amplification targeting ALT repeats. Type 3 C. albicans constituted the majority of isolates in any genotypes (66.3% for genotype A, 76.3% for genotype B and 73.4% for genotype C). Each C. albicans type showed several amplification patterns, indicating the existence of subtypes. RFLP analysis revealed that restriction profiles of PCR products corresponded to amplification patterns from PCR. CONCLUSION: The present results indicate that PCR amplifications targeting 25S rDNA and ALT repeats are useful for rapid genotyping and distinction of C. albicans involved in superficial candidiasis.

Candida albicans↗

PubMed2005

Glucose-6-phosphate dehydrogenase (G6PD) mutations in Cambodia: G6PD Viangchan (871G>A) is the most common variant in the Cambodian population.

We conducted a survey of malaria diagnoses and glucose-6-phosphate dehydrogenase (G6PD) testing in remote areas of Cambodia. Blood specimens from 670 people were collected by the finger-prick method. Of these people, 24.9% were found to have malaria, and 7.0% of people were G6PD deficient. In the Khmer, the largest ethnical population in Cambodia, the G6PD deficiency rate of males was 12.6% (25/199) whereas the rates in the minorities of the Tum Pun and the Cha Ray were 1.1% (1/93) and 3.2% (2/63), respectively. Of the G6PD-deficient subjects, 97.9% (46/47) were G6PD Viangchan (871G>A), and only one case (2.1%) was G6PD Union (1360C>T). Since G6PD Mahidol (487G>A) is common in Myanmar according to our previous study, the current finding suggests that the Cambodian population is derived from homogeneous ancestries and is different from the Myanmar population. All G6PD Viangchan cases were linked to two other mutations of 1311C>T and IVS-11 nt93T>C in the G6PD gene.

Cambodia↗

PubMed2005

Rapid and specific identification of Sporothrix schenckii by PCR targeting the DNA topoisomerase II gene.

BACKGROUND: In order to study the genotype variation of Sporothrix schenckii, we previously analyzed the genomic sequences of the DNA topoisomerase II genes of this fungus and S. schenckii-related species, such as S. schenckii var. luriei and Ceratocystis stenoceras. OBJECTIVE: To develop a PCR-based identification system that can distinguish S. schenckii from its related species for clinical diagnosis of sporotrichosis. METHODS: Based on the nucleotide sequences of the DNA topoisomerase II genes of S. schenckii, S. schenckii var. luriei and C. stenoceras, three gene-specific primers (SSHF31 and SSHR97 for S. schenckii, and SSLF64 for S. schenckii var. luriei) were designed and evaluated for their specificities in PCR amplifications. RESULTS: The primer set SSHF31/SSHR97 amplified PCR products of 663-817 bp from S. schenckii and approximately 660 bp from S. schenckii var. luriei, while SSLF64/SSHR97 exclusively amplified a major product of 305 bp from S. schenckii var. luriei. CONCLUSION: PCR targeting the DNA topoisomerase II gene specifically and rapidly distinguished S. schenckii from its related species.

DNA↗

PubMed2005

Rapid identification of Candida albicans and its related species Candida stellatoidea and Candida dubliniensis by a single PCR amplification using primers specific for the repetitive sequence (RPS) of Candida albicans.

BACKGROUND: Candidiasis is caused by several Candida species, of which Candida stellatoidea and C. dubliniensis are phenotypically close to C. albicans. Although current molecular biology-based techniques can distinguish between C. albicans and C. dubliniensis, a convenient tool that can distinguish C. stellatoidea from C. albicans has not yet been developed. OBJECTIVE: To develop a system that can simply, rapidly and specifically distinguish C. albicans from the related Candida species C. stellatoidea and C. dubliniensis. MATERIALS: Genomic DNAs were purified from various yeast species and amplified by primers specific for the repetitive sequence (RPS) of C. albicans. The PCR products were purified and sequenced in order to test the specificity of the PCR amplification. RESULTS: The PCR primers only amplified several products from C. albicans, C. stellatoidea and C. dubliniensis. Sequence analysis of the products revealed that C. stellatoidea and C. dubliniensis both had RPSs including alt repeats, similar to C. albicans. After the PCR amplification, each of the three Candida species showed a unique amplification profile. Furthermore, RFLP analysis of the PCR products using EcoRI and ClaI produced species-specific restriction profiles. CONCLUSIONS: This PCR-based technique targeting the alt repeats in the RPS is useful as a tool for the rapid identification and distinction of C. albicans, C. stellatoidea and C. dubliniensis.

Base Sequence↗

PubMed2004

Epidemiological study of Candida species in cutaneous candidiasis based on PCR using a primer mix specific for the DNA topoisomerase II gene.

BACKGROUND: We have previously reported a PCR-based identification system for pathogenic fungi by targeting the DNA topoisomerase II gene, in which primer mixes specific for this gene were used for the PCR amplifications. OBJECTIVE: To test the potential of the PCR using primer mix that is specific for the DNA topoisomerase II gene and are designated as PsVIc, for rapid identification of Candida species involved in cutaneous candidiasis, and to define the relation between Candida species and the infection lesion. METHODS: Scales from 48 patients with cutaneous candidiasis were cultured on GYEP agar plates, and the genomic DNAs were purified from the colonies and used as DNA templates for PCR amplifications. Candida was identified as individual species based on the sizes of the PCR products generated in the PCR amplifications using PsVlc. RESULTS: Four Candida species (five genotypes; Candida albicans, Candida glabrata, Candida parapsilosis I, Candida parapsilosis II and Candida tropicalis II) were identified in the patients' scales. In 19 of the patients (39.6%), multiple PCR products (two or three bands) were amplified in a DNA sample, especially derived from scales at the groin of bed-ridden older patients using napkins. CONCLUSION: The PCR-based identification using the primer mix was useful for an epidemiological study of Candida species in cutaneous candidiasis.

Adult↗

PubMed2004

Compaction and multiple chain assembly of DNA with the cationic polymer poly(aluminum chloride) (PAC).

Assembly of DNA molecules by the addition of poly(aluminum chloride) (PAC) was studied. In the absence of PAC, electron microscopy indicated the formation of elongated coiled DNA molecules. In the presence of PAC, multiple doughnut-like structures, 8-15 nm thick, formed and fused together. When salt was added, the doughnut-like structures tended to be thinner and the morphology of the fused doughnuts became irregular. We obtained a view of a single DNA structure by fluorescent microscopy, which revealed that individual DNA molecules undergo a discrete transition from an elongated to compacted state with an increase in PAC concentration. Electron microscopic observation showed that a regular doughnut is the typical structure seen under low salt conditions. At high salt concentrations, the doughnut shape deformed, yielding results similar to those produced by the salt effect on DNA assembly at high DNA concentrations.

Aluminum Chloride↗

PubMed2004

PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis.

BACKGROUND: We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. OBJECTIVE: To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR-RFLP methods for stability and reproducibility. METHODS: Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). RESULTS: In this study, six dermatophyte species (T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR-RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. CONCLUSION: The PCR and PCR-RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.

Adolescent↗

PubMed2003

Enantioseparation of imazalil residue in orange by capillary electrophoresis with 2-hydroxypropyl-beta-cyclodextrin as a chiral selector.

Chiral resolution of imazalil, a fungicide, was performed by capillary electrophoresis (CE) using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Factors affecting the chiral resolution and migration time of imazalil were studied. The optimum running conditions were found to be 5 mM ammonium dihydrogenphosphate-50 mM phosphate buffer (pH 3.0) containing 4 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of +25 kV at 20 degrees C using direct detection at 200 nm. Under these conditions, the resolution (Rs) of racemic imazalil was approximately 6. The extraction of imazalil from orange samples was done with acetonitrile under basic conditions. The extract was purified with a solid-phase extraction cartridge (Sep-Pak plus PS-2) and was analyzed by the above CE method. Eight orange samples were analyzed, and imazalil was detected in seven samples. In four of these seven oranges, the level of (-)-imazalil was the same as that of (+)-imazalil, but in the other three oranges, the level of (-)-imazalil was found to be lower than that of (+)-imazalil, suggesting that (-)-imazalil was degraded more quickly than (+)-imazalil in oranges.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2003

PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes.

BACKGROUND: We have determined nucleotide sequences of the DNA topoisomerase II genes of the dermatophyte species, and conducted a PCR-based identification system using species-specific primers for the nucleotide sequences. OBJECTIVE: To identify the major dermatophytes, Trichophyton rubrum, T. mentagrophytes, T. violaceum, M. gypseum, M. canis and E. floccosum, by PCR amplifications at the species level, without determining the nucleotide sequence. METHODS: For PCR-based identification of the major dermatophyte species, a common primer set (dPsD1) for these species and species-specific primer sets (PsT and PsME) for each species were designed based on the genomic sequences of the DNA topoisomerase II genes of the dermatophytes, and tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by the common primer set, followed by a second PCR with the primer sets consisting of species-specific primers for each dermatophyte species. RESULTS: Using dPsD1, a DNA fragment of 3390 bp was amplified from the genomic DNA of all the dermatophyte species. In the subsequent nested PCR using species-specific primer sets (PsT and PsME), both sets amplified unique sizes of PCR products, all of which corresponded to a species of the dermatophytes even in the presence of other fungal DNA. CONCLUSION: We demonstrate that the PCR-based identification targeting the DNA topoisomerase II gene is rapid and simple, and is available as a tool for the identification of the major dermatophyte species.

Arthrodermataceae↗

PubMed2003

Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes.

BACKGROUND: We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. OBJECTIVE: To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence. METHODS: The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed. RESULTS: Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum. CONCLUSION: PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.

DNA Topoisomerases, Type II↗

PubMed2003

Ascorbic acid induces a marked conformational change in long duplex DNA.

Ascorbic acid is often regarded as an antioxidant in vivo, where it protects against cancer by scavenging DNA-damaging reactive oxygen species. However, the detailed mechanism of the action of ascorbic acid on genetic DNA is still unclear. We examined the effect of ascorbic acid on the higher-order structure of DNA through real-time observation by fluorescence microscopy. We found that ascorbic acid generates a pearling structure in single giant DNA molecules, with elongated and compact regions coexisting along a molecular chain. Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation. A possible mechanism for the induction of this conformational change is discussed in relation to the interplay between the higher-order and second-order structures of DNA.

Ascorbic Acid↗

PubMed2003

Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents.

Catalase-deficient strains of the human pathogenic yeast Candida albicans were constructed using the URA-blaster method. The disruptant was viable and grew normally in an ordinary culture condition, but became extremely sensitive to treatment with hydrogen peroxide. No catalase activity was observed in a catalase (CCT)-gene-disrupted strain, 1F5-4-1, suggesting that there were no other catalase or catalase-like enzymes in this yeast. The disruptant was shown to be sensitive to higher temperature and to low concentrations of SDS, NP-40, or Triton X-100. After a wild-type CCT gene was reintroduced into the disruptant, catalase activity was restored and the strain became moderately sensitive to treatment with hydrogen peroxide. However, neither the temperature sensitivity nor the susceptibility to SDS observed in the disruptant was restored in the CCT-reintroduced strain. A model infection experiment using wild-type and dCCT strains showed that the disruptants disappeared more rapidly than the wild-type strain in mouse liver, lung, and spleen. These results suggest that the catalase plays a significant role in survival in the host immune system and thus leads this organism to establish infection in the host.

Animals↗

PubMed2003

Improvements of PCR-based identification targeting the DNA topoisomerase II gene to determine major species of the opportunistic fungi Candida and Aspergillus fumigatus.

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.

Aspergillus fumigatus↗

PubMed2003

[Atopic dermatitis and Malassezia species: a study of antigenic components of Malassezia species for immunoglobulin E of patients with atopic dermatitis].

Antigenic components extracted by treatment with beta-mercaptoethanol from M. furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis. CBB staining and lectin blots of the extracts showed that each Malassezia species contained species-dependent components at the protein level. In a Western blot with the 2-ME extracts,IgE antibodies against the Malassezia species were found in sera of 83% (for M. globosa), 74% (for M. sympodialis), 65% (for M. furfur), 56% (for M. restricta) and 50% (for M. slooffiae) of the AD patients. In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patients IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level. A major component of M. globosa was isolated from the 2-ME extract of this fungus by ion-exchange column chromatography and was referred to as Malg46b. Dot blot with the Malg46b containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of those who were positive for IgE antibodies to the 2-ME extract of M. globosa in Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in Western blot (r=0.763) The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. These results indicate that a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.

Antibodies, Fungal↗

PubMed2002

PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes.

For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.

Animals↗