JPH0824599B2 - Pathogenic fungal gene amplification primer - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting ribosomal RNA gene (DNA) of pathogenic filamentous fungi. The present invention also relates to a detection kit for detecting the gene.

[0002]

2. Description of the Related Art The number of immunocompromised patients has been increasing with the spread of advanced medical care. Deep (systemic) mycosis such as aspergillosis has become a medical problem as an important infectious disease that predominantly affects immunocompromised patients. However, deep mycosis, especially aspergillosis which is a filamentous fungal infection, is extremely difficult to diagnose, and even today, when various antifungal agents have been developed, effective diagnostic methods have not yet been established, so many The current situation is that these patients die without treatment. Diagnosis of aspergillosis is carried out by culture identification from clinical specimens, but even if pathologically clear Aspergillus infection is observed, the culture positive rate of Aspergillus is low and culture was possible. However, it usually takes two weeks or more before the formation of conidia, which is the basis for identification of Aspergillus, which makes prenatal diagnosis of aspergillosis difficult. In addition, there is no one that has been approved as a diagnostic kit for aspergillosis so far, and as a laboratory diagnostic method, a method for immunologically detecting an Aspergillus antigen or antibody, a bacterial cell component or a metabolite of a pathogenic fungus is used. Although there are biochemical detection methods, all of them have the drawbacks of low sensitivity and significantly high values even in patients with pathologically no fungal infection (including healthy people). is there. Therefore, it is required to establish a diagnostic method that is more specific to aspergillosis and that is rapid.

[0003]

The PCR method is the most sensitive gene detection method at present, which enables rapid and convenient amplification of an arbitrary gene by using a specific primer pair. The characteristics of pathogenic fungi are defined by their genes. Therefore, the presence of the pathogenic filamentous fungus can be determined by carrying out a PCR method using a primer pair corresponding to the gene region characteristic of the pathogenic filamentous fungus and detecting the gene characteristic of the pathogenic filamentous fungus. it can.
The present inventors have found that a ribosomal RNA gene (DNA) in which a sequence specific to a pathogenic filamentous fungus of the genus Aspergillus or Penicillium is repeatedly present in a pathogenic filamentous fungal gene.
I found it inside. Therefore, the presence of a pathogenic filamentous fungus can be easily determined if the gene can be detected with high sensitivity and speed by the PCR method using a sequence specific to the pathogenic filamentous fungus in the ribosomal RNA gene as a primer pair. That is, an object of the present invention is to provide a method for detecting a ribosomal RNA gene characteristic of a pathogenic filamentous fungus in a sample and a kit used therefor.

[0004]

Means for Solving the Problems To summarize the present invention, the first invention relates to a method for detecting a pathogenic filamentous fungus, which uses synthetic primer pairs represented by SEQ ID NOs: 1 and 2 in the sequence listing,
It is characterized in that a pathogenic filamentous fungal ribosomal RNA gene of the genus Aspergillus or the genus Penicillium is amplified by the PCR method and detected. A second invention relates to a kit for detecting a pathogenic filamentous fungus, which is a detection kit for detecting the pathogenic fungus using the method of the first invention, which comprises a ribosomal RNA gene of the pathogenic filamentous fungus. It is characterized by comprising the above-mentioned synthetic primer pair for amplification by the PCR method and a probe for detecting the amplified DNA.

The sequences of primer pairs characteristic of pathogenic filamentous fungi in the ribosomal RNA gene can be determined by the following procedure. (1) Of the gene sequences registered in the gene sequence data bank (GenBank), Aspergillus, various other pathogenic fungi, human and Escherichia coli 18S ribosomal RN
The A gene (DNA) is compared between the respective nucleotide sequences and is characteristic of Aspergillus fumigatus (registration number M55626) and Penicillium notatum (registration number M55628), and human (registration number M10098), E. coli (registration number M24996). ) And various pathogenic fungi shown in Table 1 are limited.

Table 1 Candida albicans M60302 Candida guilliermondi M60304 Candida kefir M60303 Candida cruzei M60305 Candida luciferani M60307 Candida parapsilosis M60307 Candida viswanatii M6011 serrata M6011 sj Cryptococcus neoformans M55625

(2) Only the 18S ribosomal RNA gene common to Aspergillus fumigatus and Penicillium notatum can be specifically amplified from the above sequence region, and human, Escherichia coli, and other pathogenic fungi shown in Table 1 can be amplified. For the gene (1), the primer pair DNA sequence (SEQ ID NO: 1 and 2 in the sequence listing) which is not amplified is determined. The primer can be synthesized as an oligonucleotide DNA by a DNA synthesizer and can be purified by HPLC.

The pathogenic filamentous fungal DNA to be detected is blood, etc.
It can be prepared from a pathogenic filamentous fungal infected sample. PC
For the R method, Taq Polymera (Taq polymera
se) gene amplification kits and automatic gene amplification devices are commercially available, using this and the primer pair of the present invention,
An amplification reaction of pathogenic filamentous fungal DNA can be performed. In the PCR method, as an enzyme, for example, thermostable tack
The process of denaturation of DNA (94 ° C) using a polymerase,
Only the target gene can be amplified exponentially by repeating the temperature cycle consisting of the process of annealing the primer DNA (55 ° C.) and the process of enzymatic synthesis of the DNA complementary strand (72 ° C.) any number of times. For example, if the temperature cycle is repeated 25 times, the target DNA is amplified about 100,000 times. This PCR method is currently the most effective method for detecting DNA with higher sensitivity than a trace amount sample. The detection of the ribosomal RNA gene after amplification can be carried out using, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, spot method, Southern blotting method and the like. In the spot method and the Southern blotting method, a specific DNA sequence in the amplification region may be selected as a probe. One example is probe 1 of SEQ ID NO: 3 in the sequence listing.

The probe DNA is the above-mentioned primer DNA.
It can be synthesized and purified in the same manner as in. Labeling the probe DNA enables highly sensitive detection. The labeling method is not limited to the radioisotope labeling method, and any known method such as an enzyme label including an avidin / biotin label and a fluorescent labeling method including a chemiprobe label may be used. The ribosomal RNA gene of the pathogenic fungus can be subjected to PCR reaction using the primers actually selected, and can be detected with high sensitivity by electrophoresis, Southern hybridization, or the like.

Thus, the ribosomal RN of the pathogenic filamentous fungus
The pathogenic filamentous fungus can be detected with high sensitivity by the PCR method using a primer pair designed from the nucleotide sequence unique to the A gene. Further, according to the present invention, a kit for preparing a pair of primers for amplifying a ribosomal RNA gene region of a pathogenic filamentous fungus and a probe for detecting the amplified DNA region is prepared as a kit, thereby facilitating the detection of the pathogenic filamentous fungus. It can be carried out. The reagent used in the kit may be in the form of a solution, a freeze-dried product, or a state immobilized on a plate or the like. As described above, the present invention enables early diagnosis of pathogenic filamentous fungal disease, leading to early treatment thereof.

[0011]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Detection of ribosomal RNA gene of Aspergillus fumigatus JCM1738 by PCR method (1) Synthesis and purification of primer (DNA) and probe (DNA) Primers (DNA) shown in SEQ ID NOs: 1 to 3 in Sequence Listing
Also, the synthesis and purification of the probe (DNA) was requested to Kurabo Industries (Kurashiki Spinning Co., Ltd.) and Biomedical Department. (2) Preparation of Aspergillus fumigatus JCM1738 Genomic DNA The culture of Aspergillus fumigatus and the preparation of genomic DNA were carried out by the method of Bainbridge et al. [Bainbridge BW et.al.
MS Microbiology Letters 66: 113-118, 1990.], and finally about 25 μg of Genome D from 1.5 ml of culture solution.
I got NA. (3) Preparation of genomic DNA of various strains of pathogenic fungi Cultivation of various strains of pathogenic fungi shown in Table 2, preparation of spheroplasts and preparation of genomic DNA are carried out by the method of Abe et al. [November 10, 1987] , Soft Science Co., Ltd., Yoshiyuki Kuchino et al., 2 persons, “Gene / protein / experimental operation blotting method” (first edition)], and finally about 100 μg of genomic DNA was obtained from 15 ml of the culture solution.

Table 2 Candida albicans TIMMM1623 Candida albicans TIMMM2726 Candida albicans (Stellatoidea) TIMMM0310 Candida guillie Mondii TIMMM0260 Candida kefir TIMIMIM TIMIMIMO03 TIMIMPIM TIMIMIPA TIMIMPIMO TIMIMPIKA TIMADAPIMO Anomala JCM3585 Saccharomyces cerevisiae TIMMM0925 Cryptococcus neoformans TIMMM0354 Cryptococcus neoformans No. 0061

(4) Aspergillus fumigatus JCM
P in a region specific to the ribosomal RNA gene of 1738
Amplification by CR method Aspergillus prepared in Examples 1- (2) and (3)
10 ng of Fumigatatus JCM1738 DNA, 10 ng of each of various strains of pathogenic fungi shown in Table 2, 1 μg of human DNA prepared by a known method, 1 μg of bovine DNA
10 ng of E. coli and 10 ng of E. coli DNA was placed in a 0.5 ml tube, and 10 μl of 10 × amplification buffer (100 mM)
Tris-HCl pH 8.3, 500 mM KCl, 1
5 mM MgCl ₂ , 0.01% (w / v) gelatin), 8
μl dNTP mixture (dATP, dGTP, dTT
P, dCTP; 100 mM each), 1 μl of 30 pmol / μl
Primer 1, 1 μl of 30 pmol / μl primer 2 and 0.5 μl of 5 units / μl tack polymerase were added, and sterile water was further added to make a solution of 100 μl. After adding 100 μl of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplifier thermal cycler. The reaction conditions are: denaturation at 94 ° C. for 5 minutes, denaturation at 94 ° C. for 1 minute → priming annealing at 55 ° C. for 2 minutes → synthesis reaction cycle at 72 ° C. for 1 minute, 25 cycles, then 72 An extension reaction was performed at 10 ° C for 10 minutes. After the reaction, take 5 μl of the lower layer reaction solution and 1.2%
Agarose gel electrophoresis was performed and the DNA was stained with ethidium bromide to confirm the amplified DNA. As a result, a single band of 385 bp was confirmed only when Aspergillus fumigatus JCM1738 was used as a template. No amplification was observed when other DNA was used as a template. (5) Detection of Aspergillus fumigatus ribosomal RNA gene amplified by PCR method by Southern hybridization 1.2% agarose gel electrophoresed with the PCR reaction solution described in Example 1- (4) Then, Southern blot was performed overnight on a nylon membrane (Amersham Highbond-N). UV transilluminator (2
The DNA was immobilized on the membrane by irradiating with a membrane (54 nm) for 10 minutes. DNA detection from this membrane is based on ECL 3
It was carried out as follows by fluorescence emission using an oligo-labeling / detection system (Amersham Japan). Prehybridization was carried out for 30 minutes at 37 ° C. in 5 ml of prehybridization buffer (0.5% blocking solution, 5 × SSC, 0.1% SDS, 5% dextran sulfate and 100 μg / ml salmon sperm DNA). Next, probe 1 tailed with fluorescein-modified dUTP using terminal transferase
Was added and hybridization was carried out at 37 ° C. overnight. After hybridization, the membrane was washed in Wash Buffer 1 (1 × SSC / 0.1% SDS) for 15 minutes at 60 ° C. with shaking, followed by Wash Buffer 2 (0.5 × SSC / 0.1%). % SDS) for 15 minutes at 60 ° C. with shaking twice. Further, wash the membrane in buffer 1 for 1 minute, then at room temperature for 6 minutes.
Shaking was performed in 0.5% blocking solution for 0 minutes.
Then, incubation was carried out at room temperature for 60 minutes in an HRP-labeled anti-fluorescein antibody solution diluted 1000-fold with buffer 2. After drying the membrane,
Hyper Film-ECL (Amersham Japan)
Exposure was carried out for 40 minutes in a cassette containing. As a result, PCR amplified DNA of Aspergillus fumigatus
, A band appeared after hybridizing with probe 1, but no band was observed in other samples.
This means that only the Aspergillus fumigatus DNA was specifically amplified with primer 1 and primer 2,
It shows that a sequence specific to the ribosomal RNA gene of Aspergillus fumigatus can be detected by hybridization with probe 1.

Example 2 PC of ribosomal RNA gene of various strains of pathogenic fungi
Detection by R method (1) Preparation of genomic DNA For each strain of pathogenic filamentous fungi shown in Table 3, Example 1
-Culture and genomic DNA were prepared by the method (2).

Table 3 Aspergillus fumigatus JCM1738 Aspergillus fumigatus TIMM1335 Aspergillus fumigatus No. 2005 Aspergillus fumigatus No. 2014 Aspergillus Fumigatus No. 2021 Aspergillus fumigatus No. 2022 Aspergillus terreus No. 2036 Aspergillus Flaves TIMM0057 Aspergillus Versicolor TIMM1290 Aspergillus nidulans TIMM0111 Aspergillus niger TIMM0113 Penicillium expansum TIMM1293

(2) Detection of ribosomal RNA gene by PCR using primer 1 and primer 2 10 ng of each of the above DNAs was placed in a 0.5 ml tube, and 10 μl of 10 × amplification buffer solution having the same composition as in Example 1 was prepared. , 8 μl dNTP mixture (dATP, dG
TP, dTTP, dCTP; 100 mM each), 1 μl of 3
0 pmol / μl primer 1, 1 μl of 30 pmol / μl primer 2 and 0.5 μl of 5 units / μl tack polymerase were added, and sterile water was further added to make 100 μl of solution. After adding 100 μl of mineral oil to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplifier thermal cycler. The reaction conditions are 94 ° C and 5
After denaturing for 1 minute, denaturation at 94 ° C for 1 minute → annealing of primer at 55 ° C for 2 minutes → synthesis reaction cycle at 72 ° C for 1 minute 25 cycles, followed by extension reaction at 72 ° C for 10 minutes It was After the reaction, 5 μl of the lower layer reaction solution
The collected DNA was subjected to 1.2% agarose gel electrophoresis and stained with ethidium bromide to confirm the amplified DNA. As a result, a single band of 385 bp was confirmed in all of the pathogenic filamentous fungi of 7 strains and 12 strains shown in Table 3.

Example 3 Preparation of Amplification / Detection Kit for Pathogenic Filamentous Fungal Gene A kit for amplifying / detecting a pathogenic filamentous fungal gene in a sample was prepared. As a primer for DNA amplification, a TE buffer (10 mM Tris, 1 mM EDT) was prepared so that each of primer 1 and primer 2 was a 30 pmol / μl solution.
A, pH 8.2) was dissolved in 40 μl to obtain a pathogenic filamentous fungal gene amplification primer solution (agent A). As a probe for detecting DNA, 1 μg of probe 1 was dissolved in 20 μl of TE buffer to prepare a pathogenic fungal gene detection probe solution (agent B). Agent A and agent B as a set, pathogenic filamentous fungal gene DN
It was used as the A amplification / detection kit (Table 4).

Table 4 Agent A Pathogenic filamentous fungal gene amplification primer A solution 40 μ
l (2μl × 20 times) B agent Pathogenic filamentous fungal gene detection probe B liquid 20μ
l (1 μl x 20 times)

[0020]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a highly sensitive detection method and detection kit for a pathogenic filamentous fungus in a sample by detecting the ribosomal RNA gene of the pathogenic filamentous fungus.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical sequence: NO Antisense: NO Sequence features: 1-19 E primer Sequence: ACTTTCGATGGTAGGATAG 19 SEQ ID NO: 2 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA ) Hypothetical sequence: NO Antisense: YES Sequence features: 1-19 E primer sequence: CAGAAGGAAAGGTCCAGCC 19 SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical sequence: NO Antisense: NO Sequence feature: 1-20 E probe sequence: CTGAGAAACGGCTACCACAT 20

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:67) (C12Q 1/68 C12R 1:80) (56) References JOURNAL OF MEDICAL MICROBILOGY, 36-4! (1992) (UK) P. 255-263 JOURNAL OF CLINICA L MICROBIOLOGY, 29-11! (1991) (USA) 2543-2549 JOURNAL OF CLINICA L MICROBIOLOGY, 30-5! (1992) (US) P. 1210-1215

Claims (2)

[Claims] 1. A method for amplifying and detecting a pathogenic filamentous fungal ribosomal RNA gene (DNA) of Aspergillus or Penicillium by PCR using the synthetic DNA primer pairs represented by SEQ ID NOS: 1 and 2 in the Sequence Listing. A method for detecting a pathogenic filamentous fungus of the genus Aspergillus or Penicillium. 2. A kit for detecting pathogenic filamentous fungi, which comprises the synthetic DNA primer pair according to claim 1 and a probe for detecting amplified DNA.