JP2012182997A - Method of detecting cryptococcosis causal agent and kit for detecting the same - Google Patents
Method of detecting cryptococcosis causal agent and kit for detecting the same Download PDFInfo
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Abstract
Description
本発明は、クリプトコックス症起因菌であるCryptococcus neoformansおよびCryptococcus gattiiを同時に区別して検出するための手段に関するものである。 The present invention relates to a means for simultaneously distinguishing and detecting Cryptococcus neoformans and Cryptococcus gattii which are causative agents of cryptococcosis.
クリプトコックス症は主にCryptococcus neoformansによって引き起こされる髄膜炎であるが、Cryptococcus gattiiが病原菌であることもあり、その場合は症状が重篤化することが多い。C.gattiiは熱帯地域に分布する菌であったが、カナダ・バンクーバー島での例があるように分布域が拡大している。健常人でも死に至る危険性がある本感染症の起因菌を同定することは有効な治療を行なうために重要である。 Cryptococcosis is meningitis mainly caused by Cryptococcus neoformans, but Cryptococcus gattii may be a pathogen, in which case the symptoms are often severe. C. gattii was a fungus that was distributed in the tropics, but its distribution range has expanded as in the case of Vancouver Island, Canada. It is important to identify the causative agent of this infection that can be fatal even for healthy people in order to provide effective treatment.
クリプトコックス症起因菌について、その特異的遺伝子または遺伝子産物（タンパク質等）の検出に関する技術は、例えば特許文献１などに開示されている。また、C.neoformansおよびC.gattiiにそれぞれ特異的な遺伝子領域をPCR増幅して各々の菌を検出する方法が非特許文献１に開示されている。
前記のとおり、クリプトコックス症起因菌がC.neoformansであるかC.gattiiであるかを正確に判定することは、有効な治療を行なうために重要な要件である。しかしながら、従来方法は、検体から菌のゲノムDNAを抽出し、PCR等で遺伝子を増幅させ、塩基配列を決定するなどの工程を含むため、結果を得るまでに約2日間を要した。 As described above, accurately determining whether the cryptococcosis-causing bacterium is C. neoformans or C. gattii is an important requirement for effective treatment. However, since the conventional method includes steps such as extracting bacterial genomic DNA from a specimen, amplifying the gene by PCR or the like, and determining the base sequence, it took about 2 days to obtain the result.
クリプトコックス症、特に重篤な症状を引き起こすC. gattii感染症に対する治療効果を有効なもとのするためには、できるだけ早く治療を開始することが必要であり、そのためにはクリプトコックス症起因菌がC.neoformansであるかC.gattiiであるかを、正確であることはもちろんのこと、迅速に判定することが不可欠である。 It is necessary to start treatment as soon as possible in order to have effective treatment for cryptococcosis, especially C. gattii infection that causes serious symptoms. It is indispensable to quickly determine whether or not C.neoformans or C.gattii is accurate.
本発明は、以上のとおりの事情に鑑みてなされたものであり、クリプトコックス症起因菌がC.neoformansであるかC.gattiiであるかを、正確かつ迅速に判定することを可能とする新しい手段を提供することを課題としている。 The present invention has been made in view of the circumstances as described above, and is a new that makes it possible to accurately and quickly determine whether a cryptococcosis-causing bacterium is C. neoformans or C. gattii. The problem is to provide means.
本発明は、前記の課題を解決するものとして以下を提供する。
（1）検体から抽出したDNAを、Cryptococcus noeformansおよびCryptococcus gattiiの夾膜合成遺伝子CAP59に特異的なプライマーセットを用いてPCR増幅すること、および
PCR産物にC. noeformansのCAP59遺伝子に特異的に結合するプローブとC. gattiiのCAP59遺伝子に特異的に結合するプローブを反応させることを含み、
C. noeformans特異的プローブが結合し、C. gattii特異的プローブが結合しないPCR産物が由来する検体がC. noeformans感染検体であると判定し、
C. gattii特異的プローブが結合し、C. noeformans特異的プローブが結合しないPCR産物が由来する検体をC. gattii感染検体であると判定すること、
を特徴とするクリプトコックス症起因菌の検出法。
（2）C. noeformans特異的プローブがSEQ ID NO:1のヌクレオチド配列からなるオリゴヌクレオチドである前記（1）の検出法。
（3）C. gattii特異的プローブがSEQ ID NO:2のヌクレオチド配列からなるオリゴヌクレオチドである前記（1）の検出法。
（4）夾膜合成遺伝子CAP59に特異的なプライマーセットが、SEQ ID NO:3のヌクレオチド配列からなるフォワードプライマーとSEQ ID NO:4のヌクレオチド配列からなるリバースプライマーのセットである前記（1）の検出法。
（5）検体に感染したクリプトコックス症起因菌がCryptococcus noeformansおよびCryptococcus gattiiのいずれであるかを検出するキットであって、
Cryptococcus noeformansおよびCryptococcus gattiiの夾膜合成遺伝子CAP59を特異的にPCR増幅するプライマーセット、
C. noeformansのCAP59遺伝子に特異的に結合するプローブ、および
C. gattiiのCAP59遺伝子に特異的に結合するプローブ、
を含むことを特徴とするクリプトコックス症起因菌検出キット。
（6）C. noeformans特異的プローブがSEQ ID NO:1のヌクレオチド配列からなるオリゴヌクレオチドである前記（5）の検出キット。
（7）C. gattii特異的プローブがSEQ ID NO:2のヌクレオチド配列からなるオリゴヌクレオチドである前記（5）の検出キット。
（8）夾膜合成遺伝子CAP59に特異的なプライマーセットが、SEQ ID NO:3のヌクレオチド配列からなるフォワードプライマーとSEQ ID NO:4のヌクレオチド配列からなるリバースプライマーのセットである前記（5）の検出キット。
The present invention provides the following to solve the above problems.
(1) PCR amplification of DNA extracted from the specimen using a primer set specific for the capsular synthesis gene CAP59 of Cryptococcus noeformans and Cryptococcus gattii, and
Reacting the PCR product with a probe that specifically binds to the CAP59 gene of C. noeformans and a probe that specifically binds to the CAP59 gene of C. gattii,
C. noeformans-specific probe binds and C. gattii-specific probe does not bind to the sample derived from the PCR product is determined to be a C. noeformans-infected sample,
Determining that a sample derived from a PCR product to which a C. gattii-specific probe binds and a C. noeformans-specific probe does not bind is a C. gattii-infected sample;
A method for detecting cryptococcosis-causing bacteria characterized by the above.
(2) The detection method according to the above (1), wherein the C. noeformans-specific probe is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1.
(3) The detection method according to the above (1), wherein the C. gattii specific probe is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2.
(4) The primer set specific to the capsule synthesis gene CAP59 is a set of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4 Detection method.
(5) a kit for detecting whether Cryptococcus noeformans or Cryptococcus gattii is a cryptococcosis-causing bacterium infected with a specimen,
A primer set that specifically PCR-amplifies the capsular synthesis gene CAP59 of Cryptococcus noeformans and Cryptococcus gattii,
A probe that specifically binds to the C. noeformans CAP59 gene; and
A probe that specifically binds to the CAP59 gene of C. gattii,
A kit for detecting cryptococcosis-causing bacteria, comprising:
(6) The detection kit according to (5), wherein the C. noeformans-specific probe is an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1.
(7) The detection kit according to (5), wherein the C. gattii specific probe is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2.
(8) The primer set specific to the capsular synthesis gene CAP59 is a set of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4 Detection kit.
本発明によれば、ゲノムDNAの抽出、PCRでの遺伝子増幅、塩基配列決定するなどの工程を含む従来方法が判定結果まで約２日間を要したのに対して、検体の感染菌がC.neoformansであるかC.gattiiであるかを、約4時間で判定することが可能となる。 According to the present invention, the conventional method including steps such as extraction of genomic DNA, gene amplification by PCR, and nucleotide sequencing required about 2 days until the determination result, whereas the infectious bacteria of the sample was C.I. Whether it is neoformans or C.gattii can be determined in about 4 hours.
本発明の検出法における「検体」は、例えば、ヒト、家畜等の動物由来の血液、髄液、喀痰、胃液、膣分泌物、口腔内粘液、鼻腔の塗抹サンプル、尿および糞便のような排出物等、クリプトコックス症起因が存在すると思われるあらゆる物を対象とする。また、食品、飲料水、温泉水のような環境中の水等、または空気清浄器等のフィルタなど、クリプトコックス症起因による汚染が引き起こされる可能性のある媒体全てが挙げられる。さらに、輸出入時における検疫等の動植物も検体としてその対象とする。 The “specimen” in the detection method of the present invention is, for example, blood derived from animals such as humans and domestic animals, cerebrospinal fluid, sputum, gastric juice, vaginal secretions, oral mucus, smear samples of nasal cavity, excretion such as urine and feces. Targets are all things that are thought to be caused by cryptococcosis. Also, all media that may cause contamination due to cryptococcosis, such as food, drinking water, hot water in the environment, and filters such as air purifiers, are included. Furthermore, animals and plants such as quarantine at the time of import / export are also considered as specimens.
本発明の検出方法においては、先ず、検体から抽出したDNAを、C. noeformansおよびC. gattiiの夾膜合成遺伝子CAP59に特異的なプライマーセットを用いてPCR増幅する。このようなプライマーセットは、
・CAP59遺伝子検出用フォワードプライマー
Cap59F3：5'-stacaagmarycktggtccaac-3'（SEQ ID NO:3）
・CAP59遺伝子検出用リバースプライマー
Cap59R3：5'-araagtrcttgtgctgcctcgc-3'（SEQ ID NO:4）
を例示することができる。このプライマーセット１組でCapAD-F、CapBC-V両方の結合部位がそれぞれ増幅される。
In the detection method of the present invention, DNA extracted from a specimen is first PCR-amplified using a primer set specific for the capsular synthesis gene CAP59 of C. noeformans and C. gattii. Such a primer set is
・ Forward primer for CAP59 gene detection
Cap59F3: 5'-stacaagmarycktggtccaac-3 '(SEQ ID NO: 3)
・ Reverse primer for CAP59 gene detection
Cap59R3: 5'-araagtrcttgtgctgcctcgc-3 '(SEQ ID NO: 4)
Can be illustrated. The binding sites of both CapAD-F and CapBC-V are amplified by one primer set.
また、PCR反応は、使用するPCR試薬のプロトコール等に従って定法による行なうことができる。 Moreover, PCR reaction can be performed by a conventional method according to the protocol of the PCR reagent to be used.
本発明方法では、このPCR反応時にC. noeformansのCAP59遺伝子に特異的に結合するプローブとC. gattiiのCAP59遺伝子に特異的に結合するプローブを用いて、PCR産物が由来する検体がC. noeformans感染検体であるかC. gatti感染検体であるかを検出する。 In the method of the present invention, a sample from which a PCR product is derived is obtained using a probe that specifically binds to the CAP59 gene of C. noeformans and a probe that specifically binds to the CAP59 gene of C. gattii during this PCR reaction. Detect whether it is an infected specimen or a C. gatti infected specimen.
プローブとしてはそれぞれ以下を例示することができる。
・Cryptococcus neoformans検出用プローブ：5'-accactggaaacgaca-3'（SEQ ID NO:1）
・Cryptococcus neoformans検出用プローブ：5'-atcgttccccgtgatt-3'）SEQ ID NO:2）
これらのプローブは、異なる標識を例えば5’端等に付加することが好ましい。例えば、6-FAM（6-Carboxyfluorescein）やVIC（2’-chloro-phenyl-1,4-dichloro-6-carboxyfluorescein）等の蛍光標識等である。
The following can be illustrated as a probe, respectively.
・ Cryptococcus neoformans detection probe: 5'-accactggaaacgaca-3 '(SEQ ID NO: 1)
・ Cryptococcus neoformans detection probe: 5'-atcgttccccgtgatt-3 ') SEQ ID NO: 2)
These probes preferably have different labels added to the 5 ′ end, for example. Examples thereof include fluorescent labels such as 6-FAM (6-Carboxyfluorescein) and VIC (2′-chloro-phenyl-1,4-dichloro-6-carboxyfluorescein).
またプローブは公知の化学合成法により作成することができる。前記に例示した短鎖プローブの場合には、3’端にMGB(Minor Groove Binder)構造を付加したものが好ましい。 The probe can be prepared by a known chemical synthesis method. In the case of the short chain probe exemplified above, it is preferable to add an MGB (Minor Groove Binder) structure to the 3 'end.
本発明の検出キットは、前記のプライマーセットおよび2種類のプローブの他に、PCR反応試薬等を含むもとして構成することができる。 The detection kit of the present invention can be configured to include a PCR reaction reagent and the like in addition to the primer set and the two types of probes.
以下、実施例を示して本発明の効果等について詳しく説明するが、本発明は以下の例に限定されるものではない。 Hereinafter, although an example is shown and an effect etc. of the present invention are explained in detail, the present invention is not limited to the following examples.
1．材料と方法
1-1．共試菌株
Cryptococcus neoformans TIMM 0362(serotype A)、C.neoformans TIMM 1316(serotype D)、C.neoformans TIMM 1317(serotype AD)、Cryptococcus gattii TIMM4904(serotype B)、C.gattii TIMM 1315(serotype C)、Aspergillus fumigatus TIMM 3968、Aspergillus niger TIMM 0115、Candida albicans TIMM 1768、Candida glabrata CBS 138T、Candida parapsilosis ATCC 90018、Rhodotorula minuta TIMM 6222、Sporobolomyces koalae JCM 15063T、Trichosporon asahii JCM 2466T、国内動物園のコアラおよびコアラ舎から分離したC.neoformans 56株、C.gattii 3株、その他のCryptococcus spp. 54株、Basidiomycetous yeasts（Cryptococcus spp.を除く）121株、ミカファンギン耐性Ascomycetous yeasts 25株。
1． Materials and methods
1-1. Co-test strain
Cryptococcus neoformans TIMM 0362 (serotype A), C. neoformans TIMM 1316 (serotype D), C. neoformans TIMM 1317 (serotype AD), Cryptococcus gattii TIMM4904 (serotype B), C. gattii TIMM 1315 (serotype C), Asperus C 3968, Aspergillus niger TIMM 0115, Candida albicans TIMM 1768, Candida glabrata CBS 138 T, was isolated from Candida parapsilosis ATCC 90018, Rhodotorula minuta TIMM 6222, Sporobolomyces koalae JCM 15063 T, Trichosporon asahii JCM 2466 T, domestic zoos Koala and Koala sha C. neoformans 56 strains, C. gattii 3 strains, other Cryptococcus spp. 54 strains, Basidiomycetous yeasts (excluding Cryptococcus spp.) 121 strains, Micafungin
1-2．染色体DNA抽出
酵母の染色体DNAの抽出にはGenとるくん・酵母用（Takara-Bio, Shiga, Japan）を使用説明書に準じて用いた。糸状菌DNAは文献（Sugita, C., K. Makimura, K. Uchida, H. Yamaguchi, and A. Nagai. 2004. PCR identification system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Med Mycol. 42: 433-437.）の記載に準じて抽出した。非培養試料はニュークリセンスDNA抽出機(BioMerieux, Marcy l'Etoile, France)の使用説明書に準じて抽出した。
1-2. Chromosomal DNA extraction For extraction of chromosomal DNA from yeast, Gen Toru-kun and yeast (Takara-Bio, Shiga, Japan) were used according to the instruction manual. Filamentous fungal DNA is available in literature (Sugita, C., K. Makimura, K. Uchida, H. Yamaguchi, and A. Nagai. 2004. PCR identification system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. Med Mycol. 42: 433-437.). The uncultured sample was extracted according to the instruction manual of Nucleic DNA extractor (BioMerieux, Marcy l'Etoile, France).
1-3．反応試薬
Eagletaq Master Mix with ROX(Roche, Basel, Switzerland)を使用説明書に準じて調製した。
1-3. Reaction reagent
Eagletaq Master Mix with ROX (Roche, Basel, Switzerland) was prepared according to the instruction manual.
1-4．使用機器
7500 Fast-Real Time PCR System(Life Technologies, California, U.S.A.)を用いた。
1-4. Used equipment
A 7500 Fast-Real Time PCR System (Life Technologies, California, USA) was used.
1-5．プローブ
・Cryptococcus neoformans検出用プローブ
CapAD-F：5'-accactggaaacgaca-3'（SEQ ID NO:1）
・Cryptococcus neoformans検出用プローブ
CapBC-V： 5'-atcgttccccgtgatt-3'）SEQ ID NO:2）
これらのプローブは、NCBIヌクレオチドデータベースのCAP59遺伝子配列に基づき設計したTaqMan MGBプローブであり、3’端にMGBが付加されている。さらに、蛍光標識としてCapAD-Fには6-FAM（6-Carboxyfluorescein）が、CapBC-VにはVIC（2’-chloro-phenyl-1,4-dichloro-6-carboxyfluorescein）がそれぞれ付加されており、異なる蛍光信号を発する。
1-5. Probes / Cryptococcus neoformans detection probes
CapAD-F: 5'-accactggaaacgaca-3 '(SEQ ID NO: 1)
・ Cryptococcus neoformans detection probe
CapBC-V: 5'-atcgttccccgtgatt-3 ') SEQ ID NO: 2)
These probes are TaqMan MGB probes designed based on the CAP59 gene sequence in the NCBI nucleotide database, and MGB is added to the 3 ′ end. Furthermore, 6-FAM (6-Carboxyfluorescein) is added to CapAD-F as a fluorescent label, and VIC (2'-chloro-phenyl-1,4-dichloro-6-carboxyfluorescein) is added to CapBC-V. Emits different fluorescence signals.
1-6．PCRプライマー
・CAP59遺伝子検出用フォワードプライマー
Cap59F3：5'-stacaagmarycktggtccaac-3'（SEQ ID NO:3）
・CAP59遺伝子検出用リバースプライマー
Cap59R3：5'-araagtrcttgtgctgcctcgc-3'（SEQ ID NO:4）
1-6. PCR primer and forward primer for CAP59 gene detection
Cap59F3: 5'-stacaagmarycktggtccaac-3 '(SEQ ID NO: 3)
・ Reverse primer for CAP59 gene detection
Cap59R3: 5'-araagtrcttgtgctgcctcgc-3 '(SEQ ID NO: 4)
1-7．リアルタイムPCR
PCR反応およびプローブ反応は、反応試薬製造者の推奨するプロトコールに従い、以下のとおりに行なった。
第1ステージ：初期変性95℃・10分、１サイクル
第2ステージ：PCR反応95℃・15秒、60℃・１分、72℃・１秒40サイクル
1-7. Real-time PCR
PCR reaction and probe reaction were performed as follows according to the protocol recommended by the reaction reagent manufacturer.
First stage: Initial denaturation 95 ° C, 10 minutes, 1 cycle Second stage: PCR reaction 95 ° C, 15 seconds, 60 ° C, 1 minute, 72 ° C, 40 seconds / cycle
2．結果
全てのC. neoformansおよびC. gattiiは、2種類のプローブおよび1種類のプライマーセットによるリアルタイムPCRによって判別された（表１）。他の菌株は2つのプローブのいずれによっても蛍光シグナルを発しなかった。また、コアラおよびコアラ舎から分離した純粋株も正確に検出された。すなわち、C.neoformans 56株はCapAD-Fプローブに陽性、CapBC-Vプローブに陰性であり、C.gattii 3株はCapBC-Vプローブに陽性、CapAD-Fプローブに陰性であり、さらに200の純粋株は両方のプローブに陰性であった。従って、誤陽性反応や擬陽性反応は存在しなかった。
2． Results All C. neoformans and C. gattii were discriminated by real-time PCR with two probes and one primer set (Table 1). Other strains did not fluoresce with either of the two probes. In addition, pure strains isolated from koalas and koala houses were also accurately detected. That is, C. neoformans 56 strain is positive for CapAD-F probe, negative for CapBC-V probe, C. gattii 3 strain is positive for CapBC-V probe, negative for CapAD-F probe, and 200 pure The strain was negative for both probes. Therefore, there was no false positive reaction or false positive reaction.
また、12頭のコアラの鼻塗抹サンプルをリアルタイムPCR直接検査および培養法によって解析した。リアルタイムPCR直接検査では、C. neoformansが3頭、C. gattiiが1頭、陽性であった（表2）。培養用ではC. neoformansが5頭、C. gattiiが1頭、陽性であった（表2）。C. neoformansは双方の結果に齟齬が認められたが、これは供試頭数が少ないこととC. neoformansは多糖を多く産生するため、PCRを阻害しやすいことに起因すると考えられる。しかしながら、C. gattiiは齟齬がなく、現在当該菌種を迅速に識別できるキットは存在しないことから、有用である。 In addition, 12 koala nasal smear samples were analyzed by real-time PCR direct inspection and culture method. In the real-time PCR direct test, 3 C. neoformans and 1 C. gattii were positive (Table 2). For culture, 5 C. neoformans and 1 C. gattii were positive (Table 2). C. neoformans showed wrinkles in both results, which is thought to be due to the small number of test specimens and the fact that C. neoformans produces a large amount of polysaccharides and thus tends to inhibit PCR. However, C. gattii is useful because it has no wrinkles and currently there is no kit that can quickly identify the species.
次に、2種類のTaqMan MGBプローブの感度を様々な濃度（5プラスミッドコピーから段階的に5×105コピー）のポジティブコントロールで試験した（図1A）。その結果、感度閾は5プラスミッドコピーであった。得られたカーブ（各濃度について3回の試験結果の平均）の傾斜から算出したCapAD-F（C. neoformans検出プローブ）およびCapBC-V（C. gattii検出プローブ）の反応効率は、それぞれ99.7%および94.9%であった（CapAD-F；sploe = -3.33、R2 = 0.999、CapBC-V；sploe = -3.45、R2 = 0.996、図1B）。両者を混合して使用した場合も、互いを妨害することはなかった。 Next, the sensitivity of the two TaqMan MGB probes was tested with positive controls at various concentrations (from 5 plasmid copies to stepped 5 × 10 5 copies) (FIG. 1A). As a result, the sensitivity threshold was 5 plasmid copies. The reaction efficiencies of CapAD-F (C. neoformans detection probe) and CapBC-V (C. gattii detection probe) calculated from the slope of the obtained curve (average of three test results for each concentration) were 99.7% each. And 94.9% (CapAD-F; sploe = −3.33, R 2 = 0.999, CapBC-V; sploe = −3.45, R 2 = 0.996, FIG. 1B). When both were used in a mixed manner, they did not interfere with each other.
CAP59遺伝子はC. neoformansゲノムの単一コピーである（Broad Institute Database, http//www.broadinstitute.org/annotation/genome/Cryptococcus_neoformans/MultHome.html）。従って、本発明方法は理論的に１試行で約５細胞を検出することが可能であることが確認された。 The CAP59 gene is a single copy of the C. neoformans genome (Broad Institute Database, http // www.broadinstitute.org / annotation / genome / Cryptococcus_neoformans / MultHome.html). Therefore, it was confirmed that the method of the present invention can theoretically detect about 5 cells in one trial.
検体の感染菌がC.neoformansであるかC.gattiiであるかを正確かつ迅速に検出することが可能となり、クリプトコックス症に対して有効な治療法を速やかに開始することが可能となる。 It is possible to accurately and quickly detect whether the infecting bacterium of the specimen is C. neoformans or C. gattii, and it is possible to quickly start an effective treatment for cryptococcosis.
Claims (8)
PCR産物にC. noeformansのCAP59遺伝子に特異的に結合するプローブとC. gattiiのCAP59遺伝子に特異的に結合するプローブを反応させることを含み、
C. noeformans特異的プローブが結合し、C. gattii特異的プローブが結合しないPCR産物が由来する検体がC. noeformans感染検体であると判定し、
C. gattii特異的プローブが結合し、C. noeformans特異的プローブが結合しないPCR産物が由来する検体をC. gattii感染検体であると判定すること、
を特徴とするクリプトコックス症起因菌の検出法。 PCR amplification of DNA extracted from the specimen using a primer set specific for the capsular synthesis gene CAP59 of Cryptococcus noeformans and Cryptococcus gattii, and
Reacting the PCR product with a probe that specifically binds to the CAP59 gene of C. noeformans and a probe that specifically binds to the CAP59 gene of C. gattii,
C. noeformans-specific probe binds and C. gattii-specific probe does not bind to the sample derived from the PCR product is determined to be a C. noeformans-infected sample,
Determining that a sample derived from a PCR product to which a C. gattii-specific probe binds and a C. noeformans-specific probe does not bind is a C. gattii-infected sample;
A method for detecting cryptococcosis-causing bacteria characterized by the above.
Cryptococcus noeformansおよびCryptococcus gattiiの夾膜合成遺伝子CAP59に特異的にPCR増幅するプライマーセット、
C. noeformansのCAP59遺伝子に特異的に結合するプローブ、および
C. gattiiのCAP59遺伝子に特異的に結合するプローブ、
を含むことを特徴とするクリプトコックス症起因菌検出キット。 A kit for detecting whether cryptococcosis-causing bacteria infected with a specimen is Cryptococcus noeformans or Cryptococcus gattii,
A primer set that specifically PCR-amplifies the capsular synthesis gene CAP59 of Cryptococcus noeformans and Cryptococcus gattii,
A probe that specifically binds to the C. noeformans CAP59 gene; and
A probe that specifically binds to the CAP59 gene of C. gattii,
A kit for detecting cryptococcosis-causing bacteria, comprising:
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