JP3167154B2 - How to detect Candida albicans - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to the serotype A of Candida albicans and
The present invention relates to a method for specifically detecting serotype B at the DNA level.

[0002]

BACKGROUND OF THE INVENTION Candida albicans is an important pathogen for immunocompromised patients. For the diagnosis of candidiasis, a serological method for detecting an antigen or an antibody has been conventionally used. However, in the antibody detection method, a decrease in antibody production ability is observed in patients with reduced immune function,
On the other hand, there is a problem as a diagnostic method because a healthy person sometimes shows a high antibody titer. On the other hand, the antigen detection method has a sensitivity problem that it cannot be detected unless at least 10 ⁵ pathogenic bacteria are present in the sample, so that a culture operation or the like of the bacteria is required. As described above, conventionally, there has been no good diagnostic method for candidiasis.

[0003]

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

[0004]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
(1) 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 at least a base sequence represented by the sequence of SEQ ID NO: 2 in the sequence listing A mixture comprising a combination of a second DNA primer containing an oligonucleotide portion of 15 bases, a DNA polymerase and an aqueous liquid test sample is subjected to a DNA amplification step, followed by (2) mitochondrial D of Candida albicans
A probe obtained by labeling a DNA fragment of about 2 kb obtained by decomposing NA with a restriction enzyme EcoO109I and having two restriction enzyme PvuII recognition sites located at about 1.3 kb apart from each other; The present invention relates to a method for detecting serotypes A and B of Candida albicans, which comprises contacting the reaction solution obtained in the step 2) with the reaction solution and performing a DNA test step for detecting a signal from the label. In the base sequence of the present specification, A is an adenine residue, C is a cytosine residue, G is a guanine residue, and T is a thymine residue.

[0005] 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 serotype A and / or serotype B of Candida albicans.
For example, blood, saliva, pharyngeal swabs, tissue sections and the like can be used.

The method for detecting Candida albicans according to the present invention mainly comprises (1) a DNA amplification step, and (2) D
It consists of an NA inspection process. In this DNA amplification step (1), PCR (Polymerase Chain Re
action) method Ru 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 one million times (Science,
239: 487-492). In the PCR method, two types of DNA primers are used: a primer for the + strand (hereinafter, referred to as a first primer) and a primer for the − strand (hereinafter, referred to as a 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 a combination of the primer and the second primer, mitochondrial DNA of Candida albicans can be obtained.
Can be specifically amplified only in a region of about 1.8 kb derived from EO3 which is one of the DNA fragments obtained by treating with EcoO109I.

[0008] The first primer and the second primer may each have a length of 15 mer to 30 mer, but are generally preferably 20 mer to 25 mer. The first primer and the second primer only need to contain an oligonucleotide of at least 15 bases out of a 25-base sequence represented by the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing, respectively. When the first primer and the second primer are less than 15 mer, the specificity during annealing decreases and non-specific binding increases. On the other hand, if it exceeds 30 mer, it is not preferable because a double strand is easily formed between primer molecules or within the molecule.
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.

Each of the first and second primers according to the present invention is prepared by a known DNA synthesis method (for example, a phosphoramidite method) using a conventional automatic DNA synthesizer (for example, manufactured by Applied Biosystems). can do.

In the DNA amplification step (1) of the present invention, the first
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, DNA capable of maintaining its activity particularly at a temperature of up to 95 ° C.
A polymerase, for example, a commercially available Taq polymerase can be used.

In the DNA amplification step of the present invention, the specific combination of the first primer and the 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 where the DNA amplification step by the PCR method can be performed. The mixture may optionally include a buffer (eg, Tris-HCl buffer),
It may contain stabilizers (eg, gelatin) or salts (eg, sodium chloride).

In the method of the present invention, P
The amplification cycle of the CR method is performed. The amplification cycle includes: (i) a DNA denaturation step (about 90 ° C.
(Ii. Seconds to about 2 minutes) (ii) Step of annealing single-stranded DNA with first and second primers (about 37 ° C. to 70 ° C. for about 30 minutes)
Second to about 3 minutes), and (iii) a DNA synthesis step using a DNA polymerase (about 65 to 80 ° C. for about 30 seconds to about 5 minutes).
The amount of DNA is doubled every cycle, and 2 ⁿ times after n cycles. In the present invention, the above-mentioned amplification cycle is repeated 10 to 60 times, preferably 20 to 40 times. In the last cycle, it is preferable to extend the heating time in step (iii) to about 5 to 10 minutes so that DNA synthesis is completely performed.

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

[0014] As the DNA testing process, Ru using the difference Than blot hybridization method.

[0015] In the Southern blot hybridization method
A non-radioactive probe (for example, an enzyme-labeled probe, a biotinylated probe, a digoxigeninated probe, or a probe labeled with a chemiluminescent substance or a fluorescent substance) can be used .

[0016] In the DNA testing process of the present invention, to use a DNA probe capable of specifically detecting Candida albicans-DNA. That is, EO3, which is a DNA fragment of about 2 kb obtained by decomposing Candida albicans mitochondrial DNA with EcoO109I, is used as a probe to specifically detect serotypes A and B of Candida albicans. Can be. This DNA fragment EO3 has two PvuII recognition sites 1.3 kb apart . This DN
The A probe EO3 is a test sample whose DNA is obtained by PCR.
Even those passing through the amplification process, it is possible to specifically recognize the Ca Nji da albicans DNA. Probe EO
As the labeling substance 3, any conventionally known substance, for example,
Radioactive substances such as ³² P, preferably non-radioactive substances (enzymes, fluorescent dyes, luminescent substances, biotin, etc.) can be used. As a method of generating a signal from the 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 can be produced in a short time by the combination of the first primer and the second primer. Amplified and synthesized specifically in large quantities. Therefore, the presence of Candida albicans in the test sample can be detected very specifically. Furthermore, 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. Further, in the method of the present invention,
Since use of specific labeled DNA probes EO 3 (the amplified was) Candida albicans-DNA, can be detected very accurately Candida albicans. Further be used in combination with the inspection process using the probe EO3 the amplification step using the first and second primers of the time required for the test is an 48 to 50 hour or so, even if carried out until Southern blot hybridization method , Get results quickly.

[0018]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples 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, Candida giramondo (C. guillierm)
ondii) M1023, Candida crusade (C.kr)
usei) M1005, Candida parapsilosis (C.
parapsilosis) M1015, Candida pseudotropicalis (C. pseudotropical)
is) M1004, Candida glabrata (C. glab)
rata) M4002, Saccharomyces cerevisiae
e) LL20 [Reference 1], Staphylococcus aureus (Staph)
ylococcus aureus) ATCC2592
3. Escherichia coli JM
109, Pseudomonas aerug
inosa) ATCC 27853, Cryptococcus neoformus (Cryptococcus neof)
ormans) NIH-68 [Reference 2], human oral squamous cell carcinoma HSC-2 cell line [JCRB cell bank] [Reference 3].

Various test Candida strains used in the following examples were prepared by Meiji Pharmaceutical University, Jatron Co., Ltd. and RIKEN (JCM: Japan Collect).
ion of Microorganisms) [References 4 and 5]. Identification of the Candida strain was performed by a conventional biological method [Reference 6] and a serological method using a polyclonal factor serum [Candida check: Jatron Co., Ltd.] [Reference 5]. The yeast was cultured at 27 ° C. in a Sabouraud medium [Difco] [Reference 4], and the bacteria were cultured at 37 ° C. in a normal nutrient liquid medium [Eiken Co., Ltd.].

Example 1: Preparation of DNA Candida albicans A serotype A (type A) strain M1012 (obtained from Meiji Pharmaceutical University) was obtained from Zymolace (Zymo Ace).
lyase) [Reference 7], and according to the method of Cryer et al. [Reference 8], total DNA is obtained.
Was prepared. That is, proteinase K (Merck &
Co) in the presence of 1% (w / v) spheroplasts
After dissolving 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). The chloroform-isoamyl alcohol extraction and RNase treatment were repeated three times,
The precipitate was precipitated with ethanol, and the resulting purified DNA 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 above was extracted with chloroform-phenol (1: 1) to remove proteins, then treated with pancreatic RNase, and crude DNA was prepared by ethanol precipitation. These were used for the hybridization and PCR described below. Cryptococcus neoformans (C. neoformans), human-derived cell lines, and the crude DNA of various bacteria were obtained from Restre.
It was prepared by the method of Po et al. [Reference 9] and the method of Maniatis et al. [Reference 10].

Example 2 Clones of Multicopy DNA Fragments
Candida albicans M101 obtained in reduction of Example 1
Total DNA of No. 2 was digested with EcoO109I and electrophoresed on a 1% agarose gel. Among the discontinuous DNA fragment bands generated on agarose, DNA was extracted from DNA fragment bands of about 0.9 kb and about 2 kb in length (each indicated by EO1 and EO3 in FIG. 1), and each of the cohesive ends was identified. The ends were blunt-ended with the Klenow fragment. Each DNA fragment was converted into pUC118
After ligation to the SmaI site of E. coli.
The plasmid was transferred to JM109 to prepare a library of plasmids having about 0.9 kb fragment and about 2 kb fragment. About 0.9
From the library of the kb insert fragment, four plasmids showing the same cleavage pattern were obtained by digestion of various restriction enzymes. These plasmids were regarded as the same clone, one representative plasmid was selected, and the insert fragment was designated as EO1. Named.
In a similar manner, three identical plasmids were obtained from a library of about 2.0 kb inserts, one of which was named EO3 (see FIG. 1).

Example 3 Labeling of DNA Probes 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
Tionallc: 3,000 Ci / mmol) and purified by a nick column (Pharmacia LKB).

Example 4 Specificity of EO3 The 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 previously treated with an alkali-treated nylon membrane (Hybond-N: Ame).
rsham), dried at 80 ° C. for 20 minutes and fixed (dot blotting). Subsequently, hybridization was performed on this membrane with the probe EO3 labeled in Example 3 at 68 ° C. for 20 minutes according to 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 -8 against an X-ray film [Fuji RX: Fuji Photo Film Co., Ltd.]
It adhered at 0 degreeC. The results are shown in FIG. The probe EO3 specifically bound to the DNA of a standard strain of Candida albicans serotypes A and B (FIG. 2; a-
1, a-2). However, six strains belonging to other medically important Candida species (such as C. tropicalis,
a-3 to b-4); cerevisiae (c-
1), St. aureus (c-2); coli
(C-3), P.E. aeruginosa (c-4), C
r. Neoforms (f-3) and human-derived cell line (f-4) did not bind to any of the standard strain DNAs. Further, probe EO3 has Candida albicans A serotypes (d-1 to e-1) and B serotypes (e-2 to e).
It bound to all the DNAs of the 10 stocks of f-2).
Table 1 shows the results of a comparative experiment of dot blot analysis using the probe EO3. 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). Probe EO1 is also specific for Candida albicans, but the binding of probe EO1 to 1.0 μg of Candida albicans DNA (lower spot in each pair) is 0.1 μg of homologous DNA
(Upper spot of each pair) was weaker than the binding of probe EO3. Probe EO1 and probe EO3
Since the labeling specific activities of the DNA fragments EO3 and
It is thought that the copy number in the genome is much higher than the fragment EO1.

Example 5: EO3 derived DNA fragment EO3 is nuclear DNA or mitochondrial DNA
(MtDNA), the total DN of several strains of Candida albicans A and B serotypes was determined.
A was cut with EcoRI and Southern blot hybridization analysis was performed using probe EO3. Ec
The oRI digestion product was electrophoresed and stained with ethidium bromide. As a result of Wills et al. [Refs. 11 and 12], five Eco-DNAs of Candida albicans mtDNA 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).
Lane 3 and lane 4). When Southern blot analysis was performed, probe EO3 showed that Candida albicans A
For both serotype and B serotype strains, two of these five bands, E2 (10.0 kb) and E3 (8.1).
kb) (Fig. 3; B). Furthermore, as a result of creating a restriction enzyme cleavage map of the DNA fragment EO3, the DNA fragment EO3 possessed two PvuII sites approximately 1.3 kb apart from each other (similar to FIG. 7 of Reference 12) (FIG. 4; A). ). From these results, it is presumed that the DNA fragment EO3 is derived from Candida albicans mtDNA.

Example 6: Synthesis of primers The partial nucleotide 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) were prepared on a DNA synthesizer. That is, 3
81A automatic DNA synthesizer (Applied Bio)
systems, Inc. ), An 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 aqueous ammonia (about 30%) was connected to the CPG column in which the synthesis process was completed, and the aqueous ammonia was extruded into the column to remove the synthesized DNA fragment. Eluted. The vial containing the collected DNA ammonia solution was sealed, heated at 55 ° C. for 6 hours, cooled to room temperature, and concentrated. The concentrate is freeze-dried, and 10 mM triethylammonium acetate (hereinafter referred to as TEA-A) (pH
7.4), and after removing the precipitate, a separation column (YMC-Pac) was applied to an L-6200 type high performance liquid chromatography apparatus (Hitachi, Ltd.) (hereinafter referred to as HPLC).
k ODS-AM313: YMC), and purification was performed using a concentration gradient of 95 mM TEA-A containing 5% acetonitrile and acetonitrile, and a main peak was collected. The obtained precipitate was suspended by adding 80% acetic acid (adjusted with acetonitrile), kept at room temperature for 30 minutes, and dried under reduced pressure. Dissolve the dried product in 10 mM TEA-A,
After extraction with diethyl ether, the extract was dried under reduced pressure. The dried DNA sample was dissolved in TEA-A, and after removing the precipitate, the second purification was performed by 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)
lmer) 0.625 U was added to the 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;
2 mM dGPT, dATP, dTTP and dCTP (P
erkin-Elmer), 250 pg of target DNA and 1 μM of each primer. DNA to PCR processor (Bio Oven: Bio Therm)
Amplification was performed by repeating 35 cycles of heat denaturation: 92 ° C. for 45 seconds, annealing (duplex formation reaction): 50 ° C. for 30 seconds, and primer extension reaction: 72 ° C. for 90 seconds. In the final cycle, the reaction was further performed at 72 ° C. for 7 minutes in order to convert the remaining product of the PCR into a complete duplex.

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

Example 8: Identification of three types of PCR products The Candida albicans serotype A and B serotype strains used in Example 7 showed slight differences in PCR products. As shown in the right end of FIG. 5A, these PCR products were classified into three types (L
Type, M type and S type). As is apparent from FIG. 5, PC of Candida albicans M1012 strain used for cloning of the DNA fragment EO3 in Example 1 above.
An M-type PCR product was obtained from the R treatment. Next, L
Type, M type and S type PCR products were converted to HincII, Pvu
Analysis was performed by digestion with II and BglII-PvuII (FIG. 6). As is clear from the restriction map (FIG. 4; B) showing the results, a short fragment containing one HincII site (indicated by hatched area a in FIG. 4B) was missing in the S-type product, and another short fragment ( BglII-H indicated by a shaded area b in FIG.
(part of the incII fragment) is missing in both the M-type and S-type products. 2 which became positive by PCR method
The same analysis was performed on the eight PCR products, and as a result, all the PCR products corresponded to any one of the three 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 portion in the BglII-HincII fragment and / or a portion including the HincII site. It is shown that it exists.

Next, in order to show that these amplified fragments were derived from the EO3 region, Southern blot hybridization using EO3 as a probe was performed on all 31 PCR-positive PCR products. As shown in Table 2 and FIG. 5B, probe EO3 bound to 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 FIG. 5A. Therefore, from these results, the amplified 1.8 kb fragment was found to be a DNA fragment EO.
3 and that the fragment is specific for all strains of Candida albicans, albeit with a slight variation in size. Was.

Example 9: Sensitivity of PCR In order to examine the sensitivity of PCR for detecting Candida albicans DNA, 10 parts of purified Candida albicans DNA was used.
A two-fold 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 by ethidium bromide staining is 2.
5 pg [corresponding to the case where 2.5 μl of a DNA (1 pg / μl) -containing solution is added to the reaction solution], whereas in Southern hybridization, 25 fg of DNA theoretically estimated to correspond to one yeast cell (25 × 10 ⁻¹⁵ g).

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

According to the PCR method using the combination of the primer (1a) and the primer (2a) according to the present invention, a region of about 1.8 kb in 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 serotype A and B serotypes, but is not present in bacteria belonging to other Candida species. Accordingly, by amplifying the region by the specific primers used in the present invention, the present invention as a DNA inspection step
When the specific probe used in the above is used, Candida albicans can be specifically detected even if only a small amount of DNA is present in the sample.

Further, Candida albicans mtDNA from DNA fragments EO 3 about 2kb of is specific against mosquitoes Nji da albicans A serotypes and B serotypes any DNA of a DNA fragment EO 3 and probe Thereby, Candida albicans can be specifically detected.

Further, the PCR method and the probe EO3
Combined with a detection limit of only 2 for purified DNA.
5 fg (DNA corresponding to one cell), which enables detection of Candida albicans with extremely high sensitivity. In addition,
The 1.8 kb fragment in DNA fragment EO3 is not present in cells of human origin. Therefore, when Candida albicans is specifically detected from a clinical material by the PCR method, there is no possibility of confusion. As described above, the present invention can provide a new method for rapid detection of Candida albicans with excellent specificity and sensitivity and rapid diagnosis of candidiasis.

The above-mentioned references are as follows. Reference 1: Szostak, JW, et al, 1979, Insertion of ag
enetic marker into the ribosomal DNA of yeast.
smid 2: 536-554 Reference 2: Kagaya, K., et al, 1985, Characterization of
pathogenic constituents of Cryptococcus neoforman
s strains. Microbiol. Immunol. 29: 517-532 Reference 3: Miyakawa, Y., et al, 1986, Production and ch.
aracterization of agglutinating monoclonal antibod
ies against predominant antigenic factors for Cand
ida albicans. J. Clin. Microbiol. 23: 881-886 Reference 4: Kagaya, K., et al, 1989, Immunologic signifi.
cance of diverse specificity of monoclonal antibod
ies against mannans of Candida albicans.J. Immuno
l. 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: a taxonomic study, 3rd ed. Elsevier Scie
nce 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: Number of nucleic acid chains: Single-stranded Sequence: ATAGGTGAGA CATATCACAG ATTAT

SEQ ID NO: 2 Sequence length: 25 Sequence type: number of nucleic acid chains: single-stranded Sequence: CATGGTTATA TCGCTAGTAT ATGAC

[Brief description of the drawings]

FIG. 1 is a photograph instead of a drawing, showing the results of agarose gel electrophoresis of a cloned DNA fragment having multiple copies derived from Candida albicans DNA. Lanes 1 and 2 are DNA size standards, respectively, lane 1 is HindIII digested λ DNA, and lane 2 is EcoT
14I-degraded λ DNA. Lane 3 is the EcoO109I digest of total DNA of Candida albicans strain M1012. Lanes 4 and 5 show the plasmid EcoRI-P having the insert fragment EO1 and the insert fragment EO3.
These are the stI digest and the EcoRI-HindIII digest.

FIG. 2 is a photograph instead of a drawing showing the result of analyzing the species specificity of a probe by dot blot hybridization, wherein (A) shows the result of probe EO3 and (B) shows the result of probe EO1. The specific activity for each probe was 1 × 10 ⁹ cpm / μg DNA. DNA from various strains was dot blotted on membranes 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: Candida giramondo (C. guillie)
rmondii) M1023, b-1: C. krusei M10
05, b-2: C. parapsilosis
losis) M1015, b-3: Candida pseudotropicalis (C. pseudo)
dotropicalis) M1004, b-4: C. glabrata
M4002, c-1: Saccharomyces cerevisiae (Sacchar
omyces cerevisiae) LL20, c-2: Staphylococcus
aureus) ATCC 25923, c-3: Escherichia coli
JM109, c-4: Pseudomonas aerug
inosa) ATCC 27853, DNA of d-1 to f-2 is composed of five strains of Candida albicans A serotype (d-1 to e-1) and five strains of Candida albicans B serotype (e-2 to f-2). ). f-3 is the DNA of Cryptococcus neoformans and f-4 is the DNA of the human cell line HSC-2.

FIG. 3 is a photograph instead of a drawing, showing the results of hybridization between EcoRI digest of Candida albicans DNA and EO3. (A) shows the results of electrophoresis of EcoRI digest of Candida albicans total DNA, and (B) shows the results of Southern blot hybridization using probe EO3. Lanes 1 and 2 are λDNAH as size standards, respectively.
IndIII digestion product and EcoT14I digestion product. Lanes 3 and 4 show Candida albicans A, respectively.
Serotype and B serotype. The positions of the individual fragments E1 to E5 which were shown to be derived from mtDNA were
Indicated according to the reported size.

FIG. 4 (A) is an explanatory view showing a restriction enzyme recognition site of a Candida albicans EO3 fragment and a partial nucleotide sequence thereof. Black lines at the left and right ends are pU
C118, and the white part in the center is the cloned fragment E
O3, and each black circle near both ends of the white portion is the position of the primer for PCR. The nucleotide sequence with an arrow (20mer) is the sequence of the PCR primer.
(B) compares the mutations in the 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, showing a 1.8 kb fragment specifically amplified by PCR from crude Candida albicans DNA. (A) shows D produced by the PCR method.
Shows the results of ethidium bromide staining of NA fragments,
(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 digest of λDNA. The left side of the DNA size standard λBstPI is the following reference strain. C. alb (A): Candida albicans A serotype, C.I. alb (B): Candida albicans B serotype, C.I. trp: Candida tropicalis (C.tropic)
alis), C.I. glm: Candida giramondi (C. guill)
iermondii), C.I. kr: C. krusei; pp: Candida parapsilosis (C. paraps)
irosis), C.I. pt: Candida pseudotropicalis (C. pse
udotropicalis), C.I. glb: Candida glabrata
a). cer: Saccharomyces cerevisiae (Sacch
aromyces cerevisiae); aur: Staphylococcus
cus aureus); coli: Escherichia coli
li), Ps. aer: Pseudomonas ae
ruginosa), and the right side of the DNA size standard λBstPI shows five conserved strains of Candida albicans serotypes A and B, respectively. L, M and S indicate the positions of the PCR products classified according to the size of the DNA.

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

FIG. 7 is a photograph, instead of a drawing, showing the results of testing the DNA detection sensitivity by PCR after amplifying Candida albicans DNA by PCR, followed by ethidium bromide staining (A) and Southern blot hybridization with probe EO3. is there. 2.5 μl of a 10-fold serial dilution series (10 ⁵ to 10 ¹ fg / μl) of the purified DNA
And TE buffer to the reaction mixture (25 μl) before adding PC
Amplified by the R method. 5 μl of each of the PCR products was subjected to electrophoresis on an agarose gel. λBst is a DNA size standard and is a BstPI digest of λDNA.

Continuation of front page (56) References Journal of Infectious Disease, Vol. 3, (1989), p. 488-494 Journal of Clinical Microbiology, Vol. 28, No. 6, (1990), p. 1204-1213 Journal of Bacterology, Vol. 164, no. 1, (1985), p. 7-13 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12Q 1/68 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (1)

(57) [Claims] (1) A first DNA primer containing an oligonucleotide portion of at least 15 bases of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing and a sequence represented by SEQ ID NO: 2 in the sequence listing A mixture comprising a combination of a second DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence, a DNA polymerase and an aqueous liquid test sample, is subjected to a DNA amplification step, and then (2) Candida It is obtained by digesting mitochondrial DNA of Albicans with restriction enzyme EcoO109I.
A probe obtained by labeling a DNA fragment of about 2 kb having two restriction enzyme PvuII recognition sites separated by kb is brought into contact with the reaction solution obtained in the DNA amplification step (1), and A. C. albicans characterized in that it is subjected to a DNA test step for detecting a signal of C. albicans
Method for detecting serotype and serotype B.