JPH0591897A - Method for detecting candida albicans - Google Patents

Abstract

PURPOSE:To specifically and quickly detect Candida albicans in high sensitivity and quickly carry out diagnosis of candidasis by amplifying DNA in a sample to be examined by a PCR method using specific two kinds of primers. CONSTITUTION:A mixed liquid containing combination of a first DNA primer containing an oligonucleotide part of at least 15 bases of a base sequence expressed by the formula 1 with a second DNA primer containing an oligonucleotide part of at least 15 bases of a base sequence expressed by formula II, a DNA polymerase and an aqueous liquid sample to be examined is passed through a DNA amplification process. Then, DNA examination of the resultant reaction liquid is carried out. The DNA examination is preferably carried out by a method using a sample obtained by decomposing a mitochondrial DNA of Candida albicans by Eco01091 and labeling a DNA fragment of 1.8kb having two sites recognized by restriction enzyme PVUII existing at a distance of 1.3kb as a probe.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for specifically detecting Candida albicans at the DNA level.

[0002]

Candida albicans is an important pathogen for immunocompromised patients. A serological method of detecting an antigen or an antibody has been conventionally used for the diagnosis of this candidiasis. However, in the antibody detection method, a decrease in antibody production ability is observed in patients with reduced immune function,
On the contrary, there is a problem as a diagnostic method because a healthy person may show a high antibody titer. On the other hand, the antigen detection method has a problem in sensitivity that it cannot be detected unless at least 10 ⁵ pathogenic bacteria are present in the sample, so that a culture operation of the bacteria is required. As described above, conventionally, there has not been a good diagnostic method for candidiasis.

[0003]

DISCLOSURE OF THE INVENTION In order to detect Candida albicans rapidly and reliably, the present inventors decomposed Candida albicans DNA using a restriction enzyme,
The obtained Candida albicans DNA fragments were cloned, and various cloned DNA fragments were evaluated as probes. As a result, they found a DNA fragment that can be used as a probe that specifically hybridizes with Candida albicans DNA. .. Next, when a partial base sequence of this probe was determined and a DNA complementary to the partial base sequence was synthesized, it was found that it can be favorably used as a primer for PCR method. The present invention is based on these findings.

[0004]

Therefore, the present invention provides a first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 1 in the sequence listing and the sequence in the sequence listing. A combination solution of a second DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence of No. 2, a DNA polymerase, and an aqueous liquid test sample is subjected to a DNA amplification step, Next, the present invention relates to a method for detecting Candida albicans, which comprises subjecting the obtained reaction solution to a DNA inspection step. In the nucleotide sequences of the present specification, A means an adenine residue, C means a cytosine residue, G means a guanine residue, and T means a thymine residue.

The liquid test sample used in the method of the present invention is not particularly limited as long as it is a sample suspected of containing Candida albicans. For example, blood, saliva, pharyngeal swab, tissue section, etc. can be used.

The method of detecting Candida albicans according to the present invention mainly comprises (1) a DNA amplification step and (2) D
NA inspection process. In this DNA amplification step (1), PCR (Polymerase Chain Re)
The action method can be used. By using the PCR method, it is possible to automatically amplify only a target DNA region from a trace amount of DNA up to about 1,000,000 times (Sc
Ience, 239: 487-491). In the PCR method, two types of DNA primers are used, a primer for the + strand (hereinafter referred to as the first primer) and a primer for the − strand (hereinafter referred to as the second primer) with the DNA region to be amplified interposed therebetween.

In the DNA amplification step (1) of the present invention, the first
By using the combination of the primer and the second primer, mitochondrial DNA of Candida albicans
It is possible to specifically amplify only a region of about 1.8 kb derived from EO3, which is one of the DNA fragments obtained by treating E.coli with EcoO109I.

The first primer and the second primer may each have a length of 15 mer to 30 mer, but it is generally preferable to have a length of 20 mer to 25 mer. The 1st primer and the 2nd primer should just contain the oligonucleotide of at least 15 bases among the sequences of 25 bases represented by the arrangement | sequence of sequence number 1 and sequence number 2, respectively. When the first primer and the second primer are less than 15 mer, the specificity upon annealing is reduced and non-specific binding is increased. Further, when it exceeds 30 mer, it becomes easy to take a double chain between or within the primer molecules, which is not preferable.
Each base constituting each of the first primer and the second primer used in the method of the present invention may be modified (for example, labeled with biotin or a luminescent substance) in any known manner.

The respective first and second primers according to the present invention are prepared by a known DNA synthesis method (for example, phosphoamidite method) using an ordinary automatic DNA synthesizer (for example, Applied Biosystems). can do.

In the DNA amplification step (1) of the present invention, the first step
The amplification cycle is repeated using a DNA polymerase, such as a thermostable DNA polymerase, with the primer and the second primer. As a thermostable polymerase, a DNA capable of maintaining its activity especially at a temperature of up to 95 ° C
Polymerases such as the commercially available Taq polymerase can be used.

In the DNA amplification step of the present invention, a specific combination of the above-mentioned first primer and second primer, DN
A mixed solution containing A polymerase and a liquid test sample is used. The amounts of the first primer, the second primer and the DNA polymerase used vary depending on the type of the liquid test sample, but can be easily determined within a range in which the DNA amplification step by the PCR method can be performed. This mixture may optionally be a buffer (eg Tris-HCl buffer),
Stabilizers (eg gelatin) or salts (eg sodium chloride) can be included.

In the method of the present invention, P
A CR method amplification cycle is performed. The amplification cycle includes (i) a denaturation step of DNA (about 90 ° C to 95 ° C, about 10
Second to about 2 minutes) (ii) Annealing step of single-stranded DNA with the first primer and the second primer (at about 37 ° C to 70 ° C, about 30 ° C)
Seconds to about 3 minutes), and (iii) a DNA synthesis step by a DNA polymerase (at about 65 ° C to 80 ° C for about 30 seconds to about 5 minutes).
DNA amount per cycle is amplified twice, after n cycles is amplified 2 ⁿ times. In the present invention, the above amplification cycle is repeated 10 to 60 times, preferably 20 to 40 times. In the last cycle, the heating time in step (iii) is preferably extended to about 5-10 minutes to ensure complete DNA synthesis.

When Candida albicans is present in the test sample, about 1.8 after the amplification cycle is completed.
A large amount of kb DNA fragment EO3 is synthesized. This DNA
A DNA fragment of about 1.8 kb, which is a part of the fragment EO3, is detected by the following DNA inspection step.

As the DNA inspection step, a method utilizing gel electrophoresis and ethidium bromide staining, a Southern blot hybrid method, or a nucleotide sequence determination method by the dideoxy method can be used. When performing the gel electrophoresis method, for example, submarine-type electrophoresis using an agarose gel as a carrier, or slab-type electrophoresis using acrylamide can be used.

Southern blot hybrid method, or in
When performing the in situ hybrid method, a non-radioactive probe (for example, an enzyme-labeled probe, a biotinylated probe, a digoxigeninized probe or a chemiluminescent substance, a probe labeled with a fluorescent substance) can be used. Furthermore, when the nucleotide sequence determination method by the dideoxy method is used, a DNA autosequencer (Applied Biosystems) using a fluorescent label can be used.

The present invention further provides Candida albicans-D.
It also provides a DNA probe that can be used for specific detection of NA. That is, EO3, which is a DNA fragment of about 2 kb obtained by decomposing mitochondrial DNA of Candida albicans with EcoO109I, is used as a probe by labeling it to specifically detect A serotype and B serotype of Candida albicans. You can This DNA fragment EO3 possesses two PvuII recognition sites located 1.3 kb apart. In addition, EO1 which is a DNA fragment of about 0.9 kb having a HincII recognition site at a position of about 0.1 kb from one end obtained by decomposing total DNA of Candida albicans with EcoO109I can be used as a probe. For these DNA probes EO3 and EO1, the test sample was PCR.
Candida albicans DNA can be specifically recognized regardless of whether it has undergone a DNA amplification step by the method or the in situ hybrid method.
As the labeling substance for the probes EO3 and EO1, any conventionally known substance, for example, a radioactive substance such as ³² P, preferably a non-radioactive substance (enzyme, fluorescent dye, luminescent substance, biotin, etc.) can be used. As a method of generating a signal from this labeling substance and further measuring the signal, any conventionally known method is used.

In the method of the present invention, only when Candida albicans is present in the test sample, a DNA fragment EO3 of about 1.8 kb is obtained in a short time by the combination of the first primer and the second primer. Is amplified and synthesized specifically in a large amount. Therefore, the presence of Candida albicans in the test sample can be detected very specifically. Further, even if the amount of Candida albicans in the test sample is very small, the DNA fragment EO3 is amplified and synthesized, so that the sensitivity is high. In the method of the present invention,
Candida albicans-DNA (optionally amplified)
Since labeled DNA probes EO3 and EO1 specific to Candida albicans are used, Candida albicans can be detected extremely accurately. Furthermore, when the amplification step using the first and second primers and the inspection step using the probe EO3 are used in combination, the time required for the inspection is about 4 to 5 hours in the case of ethidium staining, Even when it is performed, it takes about 48 to 50 hours, and the result can be obtained quickly.

[0018]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. The standard strains used in the following examples are as follows. Candida albicans M1012A serotype, Candida albicans M1445B serotype, C. tropicalis M101
7. C. guillierm
ondii) M1023, Candida krusei (C. kr)
usei) M1005, Candida parapsilosis (C.
parapsilosis) M1015, C. pseudotropical (C. pseudotropical)
is) M1004, Candida glabrata (C. glab)
rata) M4002, Saccharomyces cerevisiae
e) LL20 [Reference 1], Staphylococcus aureus (Taph)
ylococcus aureus) ATCC2592
3, Escherichia coli JM
109, Pseudomonas aerug
inosa) ATCC27853, Cryptococcus neoform
NIH-68 [Reference 2], human oral squamous cell carcinoma HSC-2 cell line [JCRB cell bank] [Reference 3].

The various Candida strains used in the following examples were Meiji Pharmaceutical University, Yatron Co., Ltd. and RIKEN (JCM: Japan Collect).
Ion of Microorganisms) [References 4 and 5]. The identification of the Candida strain was carried out by an ordinary biological method [Reference 6] and a serological method using a polyclonal factor serum [Candida Check: Yatron] [Reference 5]. Cultivation of yeast was carried out at 27 ° C. in Sabouraud medium [Difco] [Reference 4], and bacterial culture was carried out at 37 ° C. in a normal nutrient liquid medium [Eiken Co., Ltd.].

Example 1 Preparation of DNA Candida albicans A serotype (A type) M1012 strain (obtained from Meiji Pharmaceutical University) was used as a zymolyce (Zymo Ace).
lyase) to form spheroplasts [Reference 7] and then according to the method of Cryer et al. [Reference 8], total DNA
Was prepared. That is, proteinase K (Merck &
1% (w / v) spheroplast in the presence of Co)
After lysing with SDS and treating the lysate with chloroform-isoamyl alcohol (24: 1) to remove proteins, pancreatic RNase (SigmaChem.Co:5).
0 μg / ml) treatment, RNaseTI (Boehring)
erMannheim: 100 U / ml) treatment and α-amylase (Boehringer Mannheim:
0.03 U / ml). Chloroform-isoamyl alcohol extraction and RNase treatment were repeated 3 times,
Ethanol precipitated, the purified DNA obtained was collected by ethanol precipitation and dissolved in TE-buffer (10 mM Tris HCl-1 mM-Na ₂ EDTA, pH 8.0).
It was used to create a gene library. For other Candida strains, the same spheroplast lysate as described above was extracted with chloroform-phenol (1: 1) to remove proteins, and then treated with pancreatic RNase to prepare crude DNA by ethanol precipitation. These were used in the hybridization and PCR methods described below. Cryptococcus neoformans, C. neoformans, human-derived cell lines, and crude DNA of various bacteria are
It was prepared by the method of Po et al. [Reference 9] and the method of Maniatis et al. [Reference 10].

Example 2: Cloning of multicopy DNA fragments
Candida albicans M101 obtained in reduction of Example 1
Total DNA of 2 was digested with EcoO109I and electrophoresed on a 1% agarose gel. Of the discontinuous DNA fragment bands generated on agarose, DNA was extracted from DNA fragment bands having lengths of about 0.9 kb and about 2 kb (shown as EO1 and EO3 in FIG. 1, respectively), and Blunt ends were made with the Klenow fragment. PUC118 for each DNA fragment
After ligation to the SmaI site of E. coli
Transferred to JM109, a plasmid library having about 0.9 kb fragment and about 2 kb fragment was prepared. About 0.9
From the library of kb insert fragments, four plasmids showing the same cleavage pattern were obtained by digestion with various restriction enzymes. Therefore, these plasmids were regarded as the same clone, and one representative plasmid was selected and the insert fragment was designated as EO1. Named.
In the same manner, three identical plasmids were obtained from a library of about 2.0 kb insert fragment, and one insert fragment was named EO3 (see FIG. 1).

Example 3: Labeling of DNA probe EO1 (0.9 kb DNA fragment) and EO3 (2 kb DNA fragment) derived from Candida albicans were subjected to [α] using a random primer DNA labeling kit [Takara Shuzo Co., Ltd.].
- ³² P] dCTP (Amersham Interna
Tialal plc: 3,000 Ci / mmol) and purified by nick column (Pharmacia LKB).

Example 4 Specificity of EO3 DNA obtained from various strains was subjected to dot blot hybridization to examine the species specificity of the DNA fragment EO3. That is, 5 μl (0.1 μg and 1.0 μg) of each of the heat-denatured products of the crude DNA prepared in Example 1 was alkali-treated in advance with a nylon membrane (Hybond-N: Ame).
Rsham), and then dried at 80 ° C. for 20 minutes and fixed (dot blotting). Subsequently, this membrane was hybridized with the probe EO3 labeled in Example 3 at 68 ° C. for 20 minutes by the method of Maniatis et al. [Reference 10]. The specific activity of the probe was 1 × 10 ⁹ cpm / μg DNA. After hybridization, the membrane was washed, and then -8 for X-ray film [Fuji RX: Fuji Photo Film Co., Ltd.].
Adhered at 0 ° C. The results are shown in FIG. 2 and Table 1. The probe EO3 specifically bound to the standard strain DNA of Candida albicans A serotype and B serotype (FIG. 2; a- in A).
1, a-2). However, 6 strains belonging to other medically important Candida species (C. tropicalis, etc.,
a-3 to b-4), S. cerevisiae (c-
1), St. aureus (c-2), E. coli
(C-3), P. aeruginosa (c-4), C
r. It did not bind to any of the standard strain DNAs of neoformans (f-3) and human-derived cell line (f-4). Further, the probe EO3 is Candida albicans A serotype (d-1 to e-1) and B serotype (e-2 to
It bound to all the DNAs of 10 stock strains of f-2).
The results of a comparative experiment of dot blot analysis with probe EO3 are shown in Table 1. This result indicates that probe EO3 is specific for Candida albicans (including both serotypes).

Similar results were shown when EO1 was used as a probe (FIG. 2; B). The probe EO1 is also specific to Candida albicans, but the binding degree of the probe EO1 to 1.0 μg of Candida albicans DNA (the lower spot of each pair) was 0.1 μg of DNA of the same species.
It was weaker than the degree of binding of probe EO3 to (the upper spot of each pair). Probe EO1 and probe EO3
Since the labeling specific activities of DNA fragments are the same, DNA fragment EO3
It is considered that the copy number in the genome is much higher than that of the fragment EO1.

Example 5: DNA fragment derived from EO3 EO3 is nuclear DNA or mitochondrial DNA
(MtDNA), the total DN of several strains of Candida albicans A serotype and B serotype was investigated in order to determine the origin.
A was cut with EcoRI and subjected to Southern blot hybridization analysis with probe EO3. Ec
The oRI cleavage product was electrophoresed and stained with ethidium bromide. As a result, 5 Eco of Candida albicans mtDNA reported by Wills et al. [References 11 and 12] were reported.
Five discontinuous bands having a size corresponding to the size of the RI cut pieces (E1 to E5) were detected (FIG. 3; A).
Lanes 3 and 4). When Southern blot analysis was performed, probe EO3 was found to be Candida albicans A.
For both serotype and B serotype strains, two of these five bands, namely E2 (10.0 kb) and E3 (8.1).
(Fig. 3; B). Furthermore, as a result of constructing a restriction enzyme cleavage map of the DNA fragment EO3, the DNA fragment EO3 possessed two PvuII sites [similar to FIG. 7 of Reference 12] separated from each other by about 1.3 kb (FIG. 4; A). ). From these results, it is estimated that the DNA fragment EO3 is derived from Candida albicans mtDNA.

Example 6 Synthesis of Primer The partial base sequences of both end regions of the DNA fragment EO3 were determined by the dideoxy method [Reference 13] (FIG. 4; A). Based on this sequence, a pair of primers (2
0 mer) two kinds were produced by a DNA synthesizer. That is, 3
81A Automatic DNA Synthesizer (Applied Bio)
systems, Inc. ) To which the adenine CPG column was attached and primer (1a) [TAGGTGAGAC ATATCACAG
A] and primer (2a) [GTTATATCGC TAGTATATGA
] Was synthesized.

A disposable syringe (2.5 ml) containing 2.5 ml of ammonia water (about 30%) was connected to the CPG column where the synthesis process was completed, and ammonia water was pushed out into the column to remove the synthesized DNA fragment. It eluted. The vial bottle containing the recovered DNA ammonia solution was sealed, heated at 55 ° C. for 6 hours, cooled to room temperature, and then concentrated. The concentrate was freeze-dried and 10 mM triethylammonium acetate (hereinafter referred to as TEA-A) (pH
7.4), the precipitate was removed, and then a separation column (YMC-Pac) was applied to an L-6200 high performance liquid chromatography apparatus (Hitachi, Ltd.) (hereinafter referred to as HPLC).
(k ODS-AM313: YMC) was mounted and purification was performed using a concentration gradient of 95 mM-TEA-A containing 5% acetonitrile and acetonitrile, and the main peak was collected. 80% acetic acid (adjusted with acetonitrile) was added to the obtained precipitate to suspend it, and the suspension was kept at room temperature for 30 minutes and then dried under reduced pressure. Dissolve the dried product in 10 mM-TEA-A,
It was extracted with diethyl ether and dried under reduced pressure. The dried DNA sample was dissolved in TEA-A to remove the precipitate, and then purified by the second HPLC, and the main peak was collected. The purified DNA primer thus obtained was dried under reduced pressure and stored, and used in Examples described later.

Example 7: Application of EO3 to PCR method Taq DNA polymerase (heat resistance: Perkin-E
0.625 U of reaction solution 25 μl [50 mM-K
Cl, 1.5 mM MgCl ₂ , 10 mM Tris HCl (pH
8.3), 0.001% (w / v) gelatin, 0.
2 mM dGPT, dATP, dTTP and dCTP (P
erkin-Elmer), containing 250 pg of target DNA and 1 μM of each primer]. PCR processor for DNA (Bio Oven: Bio Therm)
In this, a cycle of thermal denaturation: 92 ° C. for 45 seconds, annealing (duplex formation reaction): 50 ° C. for 30 seconds, and primer extension reaction: 72 ° C. for 90 seconds was repeated 35 times for amplification. In the final cycle, the PCR product was further reacted at 72 ° C. for 7 minutes to form a complete double-stranded product.

The crude DNA obtained in Example 1 (same as shown in Table 1) was used, and after 35 cycles of PCR treatment, the PCR product was analyzed by agarose gel electrophoresis. The results are shown in FIG. 5 and Table 2. As is clear from FIG. 5, DNA of Candida albicans A serotype and B serotype out of 12 standard strains including 7 species of Candida.
The 1.8 kb fragment was detected only in the other strains, and no amplified band was observed in the other strains. Table 2 shows the results of PCR treatment of a large number of strains other than the standard strain and analysis of the PCR products on an agarose gel. Amplification of the 1.8 kb fragment was observed in all 31 strains of Candida albicans A and B serotypes. On the other hand, in the strains and human cell lines that do not belong to Candida albicans, the amplified DNA fragment was not detected at all.

Example 8: Identification of three PCR products A slight difference in the size of PCR products was found in the strains of Candida albicans A serotype and B serotype used in the above Example 7. As shown in the right end of FIG. 5A, these PCR products were selected from three types (L
Type, M type and S type). As is clear from FIG. 5, the PC of Candida albicans M1012 strain used for cloning the DNA fragment EO3 in Example 1 above.
From the R treatment, a M-type PCR product was obtained. Then L
Type, M type and S type PCR products were converted into HincII, Pvu
It was cleaved with II and BglII-PvuII and analyzed (FIG. 6). As is clear from the restriction map showing the results (FIG. 4; B), a short fragment containing one HincII site (shown by the hatched area a in FIG. 4B) was missing in the S-type product, and another short fragment ( BglII-H indicated by hatched area b in FIG.
It can be seen that part of the incII fragment) is missing in both the M and S type products. 2 became positive by PCR
As a result of performing the same analysis on the PCR products of the 8 strains, all the PCR products were in agreement with any one of the 3 types shown in FIG. These results indicate that the region involved in such size diversification is a limited region on the restriction map of the DNA fragment EO3, a part within the BglII-HincII fragment and / or a part including the HincII site. Is present in the.

Next, in order to show that these amplified fragments are derived from the EO3 region, Southern blot hybridization using EO3 as a probe was carried out on the PCR products of all 31 PCR-positive strains. As shown in Table 2 and FIG. 5B, probe EO3 bound all of the amplified 1.8 kb fragments. However, the size of the bound fragments showed size diversity corresponding to the ethidium bromide stained fragments shown in Figure 5A. Therefore, from these results, the amplified 1.8 kb fragment was the DNA fragment EO.
It was revealed that it is derived from the same region in 3 and that the fragment is contained specifically in all strains of Candida albicans, although there are slight variations in its size. It was

Example 9: Sensitivity of PCR In order to examine the sensitivity of PCR for detecting Candida albicans DNA, 10 of Candida albicans purified DNA was detected.
A double dilution series (10 ⁵ to 10 ¹ fg / μl) was prepared, 2.5 μl of each was added to 25 μl of the PCR reaction solution, and each amplification product was analyzed by agarose electrophoresis and Southern blot hybridization using EO3 as a probe. (Fig. 7). The detection limit of staining with ethidium bromide is 2.
5 pg [corresponding to the case of adding 2.5 μl of a DNA (1 pg / μl) -containing solution to the reaction solution], whereas Southern hybridization theoretically predicts that the amount of DNA is equivalent to one yeast cell of 25 fg. (25 × 10 ⁻¹⁵ g).

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

By the PCR method using the combination of the primer (1a) and the primer (2a) according to the present invention, a specific region of about 1.8 kb within a DNA region (EO3) of about 2 kb derived from Candida albicans mtDNA is specifically identified. Can be amplified. This DNA region (EO3) is specifically present in both Candida albicans A serotype and B serotype, but not in other Candida genus. Therefore, when the region of the present invention is amplified by the primer of the present invention and the probe of the present invention is preferably used in the DNA inspection step, Candida albicans can be specifically detected even if only a small amount of DNA is present in the sample. ..

Further, a DNA fragment EO3 of about 2 kb derived from Candida albicans mtDNA and a DNA fragment EO1 of about 0.9 kb derived from the total DNA of Candida albicans were isolated from both Candida albicans A serotype and B serotype, respectively. DNA fragment EO
Candida albicans can be specifically detected by using 3 or EO1 as a probe.

Further, the PCR method and the probe EO3
When combined with, the detection limit of purified DNA is only 2
Since it is 5 fg (DNA equivalent to 1 cell), Candida albicans can be detected with extremely high sensitivity. In addition,
The 1.8 kb fragment in DNA fragment EO3 is not present in human-derived cells. Therefore, there is no risk of confusion when specifically detecting Candida albicans from clinical materials by PCR. As described above, the present invention can provide a new rapid detection method for Candida albicans with excellent specificity and sensitivity and a rapid diagnostic method for candidiasis.

The references cited above are as follows: Reference 1: Szostak, JW, et al, 1979, Insertion of ag
enetic marker into the ribosomal DNA of yeast. Plas
mid 2: 536-554 Reference 2: Kagaya, K., et al, 1985, Characterization of
pathogenic constituents of Cryptococcus neoforman
Strains. Microbiol. Immunol. 29: 517-532 Reference 3: Miyakawa, Y., et al, 1986, Production and ch.
aracterization of agglutinating monoclonal antibod
ies againstpredominant antigenic factors for Candi
da albicans. J. Clin. Microbiol. 23: 881-886 Reference 4: Kagaya, K., et al, 1989, Immunologic signifi
cance of diverse specificity of monoclonal antibod
ies againstmannans of Candida albicans. J. Immunol.
143: 3353-3358 Reference 5: Meyer, S., et al, 1984, Genus 4 Candida Berk
hout, p. 585-844. In NJWKreger-van Rij (ed.), Th
e yeasts: ataxonomic study, 3rd ed. Elsevier Scien
ce Publishers BV, Amsterdam Reference 6: Kaufman, L., et al, 1986, Serodiagnosis of f
ungal diseases, p. 446-466, In NRRose, et al (e
d.), Manual of clinical laboratory immunology, 3rd
ed. American Society for Microbiology, Washingto
n, DC Reference 7: Torres-Bauza, LJ, et al, 1980, Protoplasts
from yeast and mycelial forms of Candida albican
s. J. Gen. Microbiol. 119: 341-349 Reference 8: Cryer, DR, et al, 1975, Isolation of yeast
DNA, In DM Prescott (ed.), Methods in cell biol
ogy, Vol. 12, Academic Press, New York Reference 9: Restrepo, BI, et al, 1989, Cloning of 18S
rDNAs from the pathogenic fungus Cryptococcus neof
ormans. J. Bacteriol. 171: 5596-5600 Reference 10: Maniatis, T., et al, 1982, Molecular cloni
ng: a laboratory manual. Cold Spring Harbor Labora
tory, Cold Spring Harbor, NY Reference 11: Wills, JW, et al, 1984, Repetitive DNA o
f Candida albicans: nuclear and mitochondrial comp
onents, J. Bacteriol. 157: 918-924 Reference 12: Wills, JW, et al, 1985, Circular mitocho
ndrial genome of Candida albicans contains a large
inverted duplication, J. Bacteriol. 164: 7-13 Reference 13: Sanger, F., et al, 1977, DNA sequencing wit
h chain terminating inhibitors, Proc. Natl. Acad.
Sci. USA 74: 5463-5467

[0039]

[Sequence list]

SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: ATAGGTGAGA CATATCACAG ATTAT

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: CATGGTTATA TCGCTAGTAT ATGAC

[Brief description of drawings]

FIG. 1 is a photograph instead of a drawing, which shows the result of agarose gel electrophoresis of a cloned DNA fragment having multiple copies derived from Candida albicans DNA. Lane 1 and lane 2 are DNA size standards respectively, lane 1 is HindIII degraded λDNA and lane 2 is EcoT.
14I degraded λ DNA. Lane 3 is an EcoO109I degradation product of total DNA of Candida albicans strain M1012. Lanes 4 and 5 are EcoRI-P of the plasmid with insert EO1 and insert EO3.
They are stI degradation products and EcoRI-HindIII degradation products.

FIG. 2 is a photograph instead of a drawing, showing the results of analysis of the species specificity of a probe by dot blot hybridization, (A) showing the results of probe EO3, and (B) showing the results of probe EO1. The specific activity for each probe was 1 × 10 ⁹ cpm / μg DNA. DNA from various strains was dot blotted onto the membrane in amounts of 0.1 μg (top of each pair) and 1.0 μg (bottom of each pair). The blotted DNA is as follows. a-1: Candida albicans M1012A serotype, a-2: Candida albicans M1445B serotype, a-3: Candida tropicalis (C. tropical)
is) M1017, a-4: C. guillie.
rmondi) M1023, b-1: C. krusei M10
05, b-2: C. parapsilosis (C. parapsi)
loss) M1015, b-3: Candida pseudotropicalis (C. pseu)
dotropicalis) M1004, b-4: C. glabrata
M4002, c-1: Saccharomyces cerevisiae (Sacchar
omyces cerevisiae) LL20, c-2: Staphylococcus aureus
aureus) ATCC 25923, c-3: Escherichia coli
JM109, c-4: Pseudomonas aerug
inosa) ATCC 27853, d-1 to f-2 DNA is 5 strains of Candida albicans A serotype (d-1 to e-1) and 5 strains of Candida albicans B serotype (e-2 to f-2). ) Is derived from. f-3 is Cryptococcus neoformans and f-4 is the DNA of the human cell line HSC-2.

FIG. 3 is a photograph instead of a drawing, which shows the result of hybridization of EO3 with an EcoRI degradation product of Candida albicans DNA. (A) shows the result of electrophoresis of the EcoRI degradation product of Candida albicans total DNA, and (B) shows the result of Southern blot hybridization using the probe EO3. Lanes 1 and 2 are λDNAH as size standards, respectively.
IndIII degradation product and EcoT14I degradation product, lanes 3 and 4 are Candida albicans A, respectively.
Serotype and B serotype. The positions of the individual fragments E1 to E5 shown to be derived from mtDNA are represented by Wil
It is shown according to the size reported by L. et al.

FIG. 4 (A) is an explanatory view showing a restriction enzyme recognition site of an EO3 fragment of Candida albicans and its partial nucleotide sequence. The thick black lines at the left and right ends are pU.
It is C118, and the central white part is the cloned fragment E.
O3, and each black circle near both ends of the white part is the position of the PCR primer. The nucleotide sequence with an arrow (20mer) is the sequence of the PCR primer.
(B) is a comparison of mutations in three PCR products based on the restriction enzyme analysis of the PCR products shown in FIG. The shaded area a is a region deleted in the S-type PCR product,
The shaded area b is a region deleted in the S-type and M-type PCR products.

FIG. 5 is a photograph instead of a drawing, which shows a 1.8 kb fragment specifically amplified by PCR from crude DNA of Candida albicans. (A) D produced by the PCR method
The result of ethidium bromide staining of the NA fragment is shown,
(B) shows the results of Southern blot analysis of the PCR product and the probe EO3. λBstPI is a DNA size standard and is a BstPI degradation product of λDNA. To the left of the DNA size standard λBstPI is the reference strain below. C. alb (A): Candida albicans A serotype, C.I. alb (B): Candida albicans B serotype, C.I. trp: C. tropical
alis), C.I. glm: Candida giramondi (C. guilll
iermondii), C.I. kr: C. krusei, C. pp: Candida parapsilosis (C. paraps
ilosis), C.I. pt: Candida pseudotropicalis (C. pse
Udotropicalis), C.I. glb: C. glabrata
a), S. cer: Saccharomyces cerevisiae
aromyces cerevisiae), S. aur: Staphylococcus
cus aureus), E. coli: Escherichia coli
li), Ps. aer: Pseudomonas ae
ruginosa), the right side of the DNA size standard λBstPI shows five conserved strains of Candida albicans A serotype and B serotype, respectively. L, M and S indicate the positions of PCR products classified by the size of DNA.

FIG. 6 is a photograph instead of a drawing, which shows the results of restriction enzyme analysis of three PCR products. Candida albicans A20
L strains from 7 strains (lanes 3, 6 and 9), M strains from Candida albicans M1012 strains (lanes 4, 7 and 10) and S strains from Candida albicans 8624 strains (lanes 5, 8 and 11). Each amplification product is H
It was digested with indIII (lanes 3-5), PvuII (lanes 6-8) and BglII-PvuII (lanes 9-11) and electrophoresed on an agarose gel. Lanes 1 and 2 are DNA size standards, and Lane 1 is a HindIII degradation product of λDNA,
Lane 2 is a BstPI degradation product of λDNA.

FIG. 7 is a photograph as a substitute for a drawing, which shows the results of testing the DNA detection sensitivity by PCR method by amplifying Candida albicans DNA by PCR method and then by ethidium bromide staining (A) and Southern blot hybridization with probe EO3. is there. 2.5 μl of 10-fold serial dilution series of purified DNA (10 ⁵ to 10 ¹ fg / μl)
And TE buffer to 25 μl of reaction mixture before adding PC
It was amplified by the R method. 5 μl of each PCR product was electrophoresed on an agarose gel. λBst is a DNA size standard and is a BstPI degradation product of λDNA.

Claims (3)

[Claims] 1. A first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence number 1 in the sequence listing and the base sequence represented by the sequence number 2 in the sequence listing. A second D containing an oligonucleotide portion of at least 15 bases of
Detection of Candida albicans, characterized by subjecting a mixed solution containing a combination of NA primers, DNA polymerase and an aqueous liquid test sample to a DNA amplification step, and subsequently subjecting the obtained reaction solution to a DNA inspection step Method. 2. Obtained by degrading mitochondrial DNA of Candida albicans with a restriction enzyme EcoO109I,
It is characterized in that a DNA fragment of about 1.8 kb having two restriction enzyme PvuII recognition sites present at about 1.3 kb apart is labeled, brought into contact with a test sample as a probe, and the signal from the label is detected. , Candida albicans detection method. 3. A DNA fragment of about 0.9 kb, which is obtained by digesting all the DNA of Candida albicans with a restriction enzyme EcoO109I and has a restriction enzyme HincII recognition site at a position of about 0.1 kb from one end, is used as a probe. A method for detecting Candida albicans, which comprises contacting a test sample and detecting a signal from the label.