JP2004173628A - Probe for detecting haemophilus influenzae and detection method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for specifically and quickly detecting the presence of Haemophilus influenzae taken into a specimen. <P>SOLUTION: The detection probe contains a fragment of DNA of Haemophilus influenzae, especially one or more kinds of nucleic acid fragments having a base length of 350-600 and exhibits a specific cross-reactivity with the DNA of the microorganism. <P>COPYRIGHT: (C)2004,JPO

Description

【００８１】
表１において、「Ｘ」とは、プローブ原液の濃度に応じて好ましいプローブ濃度となるように添加することができる容量を指し、この容量に伴い精製水量Ｙを決定して最終容量を調整する。
【００８２】
標識付した後、反応液にＴＥを１００μｌ 加えて反応を停止させる。 反応停止液をスピンカラムに注入し、約４℃にて、約３８０×ｇで約１０分間遠心分離して、遊離のヌクレオチドを除く。 次に、溶出液の濃度を吸光度計により測定し、ＴＥで約１０ｎｇ／μｌ に調製する。
【００８３】
標識付けを確認するために、標識付けしたプローブＨＩ−２１−８の０．５μｌをメンブレンに滴下して、風乾する。 ブロッキング試薬にこのメンブレンを浸し、室温で３０分間ブロッキングする。 ０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）と０．１５ｍｏｌ／ｌ 塩化ナトリウムで５，０００倍に希釈したアルカリフォスファターゼ標識抗ジゴキシゲニン抗体溶液に、そのメンブレンを室温で３０分間浸す。 ０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）および０．１５ｍｏｌ／ｌ 塩化ナトリウムにメンブレンを浸し、室温で約１０分間振とうして、２回洗浄する。 ０．５ｍｏｌ／ｌ トリス塩酸（ｐＨ ９．５）、０．１５ｍｏｌ／ｌ 塩化ナトリウム、５０ｍｍｏｌ／ｌ 塩化マグネシウムに、室温下で、メンブレンを約１０分間浸す。
【００８４】
室温および遮光下で、発色試薬にメンブレンを、約１０分間浸す。 メンブレンをＴＥに浸し、発色を停止させる。 スポット下部分の青紫色の発色で、標識付の確認を行う。
【００８５】
１ｍｌのディスポーザブルシリンジに、少量の滅菌済みグラスウールを充填してスピンカラムを作製する。 １ｍｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）、１ｍｍｏｌ／ｌのＥＤＴＡ、０．１％ ＳＤＳで膨潤させたセファデックスＧ−５０をシリンジに詰める。 １５ｍｌのディスポーザブルコニカルチューブにシリンジを入れ、約４℃にて、約３２０×ｇで約１０分間遠心分離し、余分の緩衝液を落とす。 ディスポーザブルコニカルチューブからシリンジを抜き、排出された緩衝液を捨てた後、１．５ｍｌのエッペンドルフ型チューブをディスポーザブルコニカルチューブの底に入れ、その上にシリンジを入れる。
【００８６】
ドットブロットハイブリダイゼーション
プローブの特異性を確認するために、以下の手順に従って、ドットブロットハイブリダイゼーションを行う。
【００８７】
まず、スポットした各ゲノムＤＮＡを変性するために、定法に従い０．５ｍｏｌ／ｌ 水酸化ナトリウム、１．５ｍｏｌ／ｌ 塩化ナトリウム溶液で飽和した濾紙（ワットマン社製３ＭＭ）上に、調製した各種細菌ゲノム１００ｎｇ をナイロンメンブレン（ポールバイオダインタイプＢ、日本ポール社製）にスポットし、風乾したメンブレンを１０分間静置する。 次に、０．５ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）、１．５ｍｏｌ／ｌ 塩化ナトリウム溶液で飽和した前出の濾紙上に１０分間静置して変性ＤＮＡを中和する。 さらに２×ＳＳＣ（Ｓｔａｎｄａｒｄ Ｓａｌｉｎｅ Ｃｉｔｒａｔｅ）溶液で飽和した前記濾紙上に５分間静置し、洗浄する。
【００８８】
そして、メンブレンを風乾し、２×ＳＳＣ溶液にメンブレンを浸し、５分間浸透する。 定法に従い、プラスチックバッグ内でプレハイブリタイゼーション溶液にメンブレンを浸し、４２℃で、６０分間親和させる。 プラスチックバッグ内でプローブ４００ｎｇを含むハイブリタイゼーション溶液の１５ｍｌにメンブレンを浸し、４２℃で、一晩反応させる。 次に、２×ＳＳＣ、０．１％ ＳＤＳ（ラウリル硫酸ナトリウム）溶液にメンブレンを浸し、５分間洗浄する（この工程を２回繰り返す）。 その後、０．１×ＳＳＣ、０．１％ＳＤＳ溶液にメンブレンを浸し、６０℃で、１０分間洗浄する（この工程を３回繰り返す）。 ２×ＳＳＣ溶液にメンブレンを浸し、５分間洗浄する。 メンブレンを３％ウシ血清アルブミン、１％ブロッキングバッファー（ベーリンガー社製）、０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）、０．１５ｍｏｌ／ｌ 塩化ナトリウムを含む溶液にメンブレンを浸し、３０分間緩慢に振とうする。
【００８９】
次いで、アルカリフォスフアターゼ標識抗ジゴキシゲニン抗体（ベーリンガー社製）を、０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）および０．１５ｍｏｌ／ｌ 塩化ナトリウム溶液で５，０００倍希釈した溶液にメンブレンを浸し、３０分間緩慢に振とうする。 次に、０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ７．５）、０．１５ｍｏｌ／ｌ 塩化ナトリウム溶液にメンブレンを浸し、１５分間振とうする（この工程を２回繰り返す）。 ０．１ｍｏｌ／ｌ トリス塩酸（ｐＨ ９．５）、０．１ｍｏｌ／ｌ 塩化ナトリウム、５ｍｍｏｌ／ｌ 塩化マグネシウムを含む溶液にメンブレンを浸し、５分間振とうする。 ＮＢＴ−ＢＣＩＰ溶液（ＧＩＢＣＯ ＢＲＬ社製）にメンブレンを浸し、遮光下で発色反応させる。 ＴＥ（１０ｍｍｏｌ／ｌ トリス塩酸（ｐＨ８．０）、１ｍｍｏｌ／ｌ ＥＤＴＡ）にメンブレンを浸し、発色反応を止め、風乾する。 プレハイブリダイゼーション溶液およびハイブリダイゼーション溶液の組成を、以下の表２に示す。
【００９０】
【表２】

【００９１】
前述したｉｎ ｓｉｔｕハイブリダイゼーション工程において使用される界面活性剤としては、公知の界面活性剤が使用できる。
【００９２】
界面活性剤は、アニオン界面活性剤、非イオン性界面活性剤、カチオン界面活性剤および両性界面活性剤に大別される。
【００９３】
アニオン界面活性剤とは、陰イオン界面活性剤とも称されるものであって、水中で電離して有機陰イオンとなるものである。 界面活性剤の分子中の親油基をＲとして表現すると、ＲＣＯＯＮａ、ＲＳＯ_３Ｎａ、ＲＳＯ_４Ｎａの式で表される。 ＲＣＯＯＮａのように弱酸性基を含有するものでは、その水溶液は加水分解しやすく弱アルカリ性を呈するが、ＲＳＯ_３Ｎａ， ＲＳＯ_４Ｎａなどの強酸性基を有するものでは、その水溶液は、加水分解を受けにくく、中性を呈する。 陰イオン性であるから、多量の陽イオン性物質の存在で界面活性を失うことがあり、また強酸性にした時にも失活する。
【００９４】
非イオン性界面活性剤とは、親水基が非イオン性のものをいう。 親水基として酸化エチレン基（−ＣＨ_２ＣＨ_２Ｏ−）が多用され、この官能基の数が多くなるに従って、親水性が増す。 反対に、親油基の炭素数が増加すると、親油性が増加する。
【００９５】
従って、親水性や親油性を様々に変化させた界面活性剤が得られるのが特徴である。 非イオン性界面活性剤は、水中で電離せず、無機塩の影響も受けにくいため、生体に及ぼす作用も少ない。 しかも、洗浄作用は、強力で、泡立ちは比較的少ないため、洗剤のみならず、医薬品、化粧品、食品などの様々な用途で使用される。 水溶性の非イオン性界面活性剤は、温度が上昇すると、ある時点で水に溶解しにくくなり、水溶液が濁り出すが、これは親水基と水との水素結合が切断されるために生じる。
【００９６】
カチオン界面活性剤は、陽イオン界面活性剤とも称され、これは、水中で、電離して有機陽イオンとなるものである。 カチオン界面活性剤は、一般に洗浄作用は大きくはないが、細菌などのアニオン性のものと強く結合するため、殺菌作用が大きい。 また、繊維やプラスチックでの帯電防止能もある。 代表的なカチオン界面活性剤であるドデシルトリメチルクロリド［Ｃ_１２Ｈ_２５（ＣＨ_３）_３Ｎ］Ｃｌは水溶性であるが、一方で、ジドデシルジメチルアンモニウムクロリド［（Ｃ_１２Ｈ_２５）_２（ＣＨ_３）_２Ｎ］Ｃｌは水に溶解しにくく、水中では２分子膜状のベシクルを形成し、これはベンゼンには溶解する。
【００９７】
両性界面活性剤とは、分子内にアニオン基とカチオン基の両者を併せ持っている界面活性剤である。 水溶液中での電離状態はアミノ酸に類似しており、両性界面活性剤には、アミノ酸誘導体が多く存在する。 従って、アミノ酸と同様に等電点を有し、等電点よりアルカリ性側にある場合にはアニオン界面活性剤として、酸性側にある場合にはカチオン界面活性剤として作用する。 等電点で水溶性は最低となり、表面張力も最も低下する。 両性界面活性剤は、殺菌剤、帯電防止剤などの用途に用いられている。
【００９８】
また、アニオン界面活性剤は、カルボン酸型、スルホン酸型、硫酸エステル型およびリン酸エステル型に分類され、非イオン性界面活性剤は、エステル型、エーテル型、エステルエーテル型およびアルカノールアミド型に分類される。 カチオン界面活性剤は、アルキルアミン塩型および第四級アンモニウム塩型に分類され、両性界面活性剤は、カルボキシベタイン型、２−アルキルイミダゾリンの誘導型およびグリシン型に分類される。
【００９９】
さらに、アニオン界面活性剤のカルボン酸型は、脂肪酸モノカルボン酸塩、Ｎ−アシルサルコシン塩およびＮ−アシルグルタミン酸塩に細分される。 それぞれの代表例として、脂肪酸モノカルボン酸塩として、ラウリン酸ナトリウムおよび薬用石鹸が、Ｎ−アシルサルコシン塩として、Ｎ−ラウロイルサルコシンナトリウム、Ｎ−アシルグルタミン酸塩、それに、Ｎ−ラウロイルグルタミン酸二ナトリウムなどがある。 また、スルホン酸型は、ジアルキルスルホコハク酸塩、アルカンスルホン酸塩、アルファオレフィンスルホン酸塩、直鎖アルキルベンゼンスルホン酸塩、アルキル（分岐鎖） ベンゼンスルホン酸塩、アルキルナフタレンスルホン酸塩、ナフタレンスルホン酸塩−ホルムアルデヒド縮合物およびＮ−メチル−Ｎ−アシルタウリン塩に細分される。 その代表例として、ジアルキルスルホコハク酸塩として、ジオクチルスルホコハク酸ナトリウム、アルカンスルホン酸塩はドデカンスルホン酸ナトリウム、直鎖アルキルベンゼンスルホン酸塩には直鎖ドデシルベンゼンスルホン酸ナトリウムが、また、アルキル（分岐鎖）ベンゼンスルホン酸塩として、ドデシルベンゼンスルホン酸ナトリウム、アルキルナフタレンスルホン酸塩として、ブチルナフタレンスルホン酸ナトリウム、Ｎ−メチル−Ｎ−アシルタウリン塩としてはＮ−メチル−Ｎ−ステアロイルタウリンナトリウムなどがある。
【０１００】
また、硫酸エステル型は、アルキル硫酸塩、ポリオキシエチレンアルキルエーテル硫酸塩および油脂硫酸エステル塩に細分される。 その代表例として、アルキル硫酸塩として、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウムおよびセチル硫酸ナトリウム、ポリオキシエチレンアルキルエーテル硫酸塩はポリオキシエチレンラウリルエーテル硫酸トリエタノールアミンなどがある。 また、リン酸エステル型は、アルキルリン酸塩、ポリオキシエチレンアルキルエーテルリン酸塩およびポリオキシエチレンアルキルフェニルエーテルリン酸塩に細分される。 その代表例として、アルキルリン酸塩として、モノラウリルリン酸二ナトリウムがある。
【０１０１】
ポリオキシエチレンアルキルエーテルリン酸塩には、リン酸ナトリウムポリオキシエチレンラウリルエーテルおよびリン酸ポリオキシエチレンオレイルエーテル（８ＭＯＬ）がある。
【０１０２】
非イオン性界面活性剤のエステル型は、脂肪酸グリセリン、脂肪酸ソルビタンおよび脂肪酸ショ糖エステルに細分される。 それぞれの代表例として、脂肪酸グリセリンとして、モノステアリン酸グリセリン、脂肪酸ソルビタンとしてはモノステアリン酸ソルビタン、トリオレイン酸ソルビタン、セスキオレイン酸ソルビタン、モノラウリン酸ソルビタン、ポリソルベート２０（ポリオキシエチレンソルビタン脂肪酸エステル）、ポリソルベート６０およびポリソルベート８０、脂肪酸ショ糖エステルはステアリン酸ショ糖エステルがある。 また、エーテル型は、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルフェニルエーテルおよびポリオキシエチレンポリオキシプロピレングリコールに細分される。 その代表例として、ポリオキシエチレンアルキルエーテルとして、ポリオキシエチレンラウリルエーテル、ポリオキシエチレンステアリルエーテルおよびポリオキシエチレンセチルエーテルなどがあり、ポリオキシエチレンアルキルフェニルエーテルとして、ポリオキシエチレンノニルフェニルエーテルおよびポリオキシエチレンオクチルフェニルエーテルがある。 また、エステルエーテル型は、脂肪酸ポリエチレングリコールおよび脂肪酸ポリオキシエチレンソルビタンに細分される。 それそれの代表例として、脂肪酸ポリエチレングリコールには、オレイン酸ポリエチレングリコールが、また、脂肪酸ポリオキシエチレンソルビタンには、パルミチン酸ポリオキシエチレンソルビタンおよびポリオキシエチレンソルビタンモノラウレートなどがある。 また、アルカノールアミド型は、脂肪酸アルカノールアミドだけであり、ラウリン酸ジエタノールアミドがその代表例である。
【０１０３】
カチオン界面活性剤のアルキルアミン塩型には、モノアルキルアミン塩、ジアルキルアミン塩およびトリアルキルアミン塩があり、モノステアリルアミン塩酸塩がその代表例である。 また、第四級アンモニウム塩型は、塩化（または臭化、沃化）アルキルトリメチルアンモニウム、塩化（または臭化、沃化）ジアルキルジメチルアンモニウム、および塩化アルキルベンザルコニウムに細分される。それぞれの代表例として、塩化（または臭化、沃化）アルキルトリメチルアンモニウムとして、塩化ステアリルトリメチルアンモニウムが、塩化（または臭化、沃化）ジアルキルジメチルアンモニウムとして、塩化ジステアリルジメチルアンモニウムが、また、塩化アルキルベンザルコニウムとして、塩化ラウリルベンザルコニウムがある。
【０１０４】
両性界面活性剤のカルボキシベタイン型には、アルキルベタインしかなく、ラウリルベタインが、その代表例である。 また、２−アルキルイミダゾリンの誘導型としては、２−アルキル−Ｎ−カルボキシメチル−Ｎ−ヒドロキシエチルイミダゾリニウムベタインだけであり、２−ウンデシル−Ｎ−カルボキシメチル−Ｎ−ヒドロキシエチルイミダゾリニウムベタインが、その代表例として挙げられる。 また、グリシン型として、アルキル（またはジアルキル）ジエチレントリアミノ酢酸があり、その代表例として、ジオクチルジエチレントリアミノ酢酸がある。
【０１０５】
さらに、上掲のものに加えて、Ｔｒｉｔｏｎ Ｘ−１００、ラウリルサルコシン、サポニン、ＢＲＩＪ３５、アルキルアリルポリエーテルアルコール、高級アルコール硫酸化物、Ｎ−ココイル−Ｌ−アルギニンエチルエステルＤＬ−ピロリドンカルボン酸塩、Ｎ−ココイル−Ｎ−メチルアミノエチルスルホン酸ナトリウム、コレステロール、自己乳化型モノステアリン酸グリセリン、スクワラン、ステアリルアルコール、ステアリン酸ポリオキシル４０、セタノール、セトマクロゴール１０００、セバシン酸ジエチル、ノニルフェノキシポリオキシエチレンエタン硫酸エステルアンモニウム、ポリオキシエチレンオレイルアミン、ポリオキシエチレンソルビットミツロウ、ポリオキシル３５ヒマシ油、マクロゴール４００、Ｎ−ヤシ油脂肪酸アシルＬ−アルギニンエチル・ＤＬ−ピロリドンカルボン酸塩、ラウリルジメチルアミンオキシド液、ラウロマクロゴール、メチルセルロース、ＣＭＣ（カルボキシメチルセルロース）、ポリオキシエチレン硬化ヒマシ油２０およびポリオキシエチレン硬化ヒマシ油６０、ＣＨＡＰＳ、デオキシコール酸、ジギトニン、ｎ−ドデシルマルトシド、ノニデットＰ４０、ｎ−オクチルグルコシド、オクチルチオグルコシド、ラウリル酸シュクロース、ドデシルポリ（エチレングリコールエーテル）ｎ，ｎ−ドデシル−Ｎ，Ｎ−ジメチル−３−アンモニオ−１−プロパンスルフォネート等も利用することができる。
【０１０６】
上掲の各種界面活性剤は、ｉｎ ｓｉｔｕハイブリダイゼーションの工程において使用されることが重要であり、その使用方法は特に限定されない。 例えば、プローブ液またはプローブ希釈液中に混合されていてもよいし、プローブ液とは別に調製した界面活性剤を含有する溶液を、プローブ液を塗抹部位に塗布する前、同時または後に添加してもよいし、当業者は適宜変更することができる。
【０１０７】
なお、本発明において、必要に応じて、陽性コントロールフローブを調製することもできる。 例えば、まず、Ｕ９３７細胞（ＡＴＣＣ ＣＲＬ−１５９３．２）のゲノムＤＮＡの抽出と精製を行うために、３７℃、５％炭酸ガスインキュベーター内で、ＲＰＭＩ１６４０培地（２５ｍｌ）を入れた細胞培養フラスコ（１７５ｃｍ^３）内でＵ９３７細胞を培養する。
【０１０８】
Ｕ９３７培養液を５０ｍｌの遠沈管に入れ、４℃にて、２２０×ｇで１０分間遠心分離し、Ｕ９３７細胞を回収する。 細胞を１０ｍｌのＰＢＳで懸濁洗浄し、再度４℃にて、１８０×ｇで１０分間遠心分離し、細胞を回収する。 その後、上清を廃棄して、細胞を１ｍｌの２００μｇ／ｍｌプロテネースＫ含有１％ＳＤＳ含有ＴＥ溶液で懸濁し、３７℃で３０分間放置する。 フェノール抽出を３〜４回繰り返し、除蛋白を行う。 エタノール沈殿によって析出したゲノムを回収し、５００μｌの２．５μｇリボヌクレアーゼ含有滅菌精製水に溶解し、４２℃で３０分間放置する。 フェノール抽出を２〜３回繰り返し、除蛋白を行う。 エタノール沈殿によって析出したゲノムを回収し、５００μｌのＴＥに溶解する。 その後、吸光度計により濃度を測定し、ジゴキシゲニンラベルに供することにより、陽性コントロールプローブを作製することができる。 また、陽性コントロールプローブは、Ｕ９３７ゲノムを１００ｎｇスポットしたメンブレンに、陽性コントロールプローブをドットハイブリダイゼーションした時に、ハイブリッド形成が確認できるものを用いるのがよい。
【０１０９】
同様に、必要に応じて、陰性コントロールプローブを公知の方法で調製することもできる。
【０１１０】
その他の実施態様
本発明の検出方法を利用したヘモフィリス インフルエンザ菌の検出および／または同定のためのキットも、本発明によって提供される。 本発明のキットによれば、ＤＮＡを得る工程で使用される酵素（ＤＮＡ露出処理剤）が、少なくとも、リゾスタフィン、リゾチーム、Ｎ−アセチルムラミダーゼ、ザイモラーゼからなるグループから選択される１種以上の酵素、界面活性剤が添加されたプローブ液、それに、１種以上の検出用ＤＮＡプローブを具備している。 本発明のキットには、以下の実施例に例示するような、血液分離試薬、酵素前処理試薬、酵素試薬、アセチル化試薬、プローブ液、ブロッキング試薬、標識抗体、標識抗体希釈液、発色前処理液−１、発色前処理液−２、発色試薬、対比染色液、ＰＢＳ原液、ハイブリダイゼーション原液、標識抗体洗浄液、発色試薬洗浄液、ＡＰＳコートスライドグラス、プローブ希釈液、バッファーＡ等を利用することが可能である。 これらの内、少なくとも、酵素試薬とプローブ液とを含むことが好ましい。 また、クロロホルム、エタノール、無水酢酸、ＤＭＳＯ、ＰＭＳＦ、ホルムアミド、酢酸、塩酸、水酸化ナトリウム等の各種試薬を用いることも可能である。 さらに、低速遠心機、恒温器、血球計算盤、振とう機、湿潤箱、恒温槽、光学顕微鏡、可変式ピペット、採血管、チップ、ピペット、染色ビン、メスシリンダー、注射筒、０．２μｍシリンジトップフィルターなどの器具を装備していてもよい。
【０１１１】
本発明によれば、生体由来の食細胞を含む臨床検体中に含まれる、食細胞によって貪食された外来微生物の遺伝子をモニターする方法が提供される。
【０１１２】
また、本発明によれば、原因菌の候補となる微生物の遺伝子を同定する工程を含み、同定された結果に基づいて敗血症原因菌または菌血症原因菌を特定する方法も提供される。 この方法によれば、様々な敗血症が疑われた患者血液の診断に実際に応用したところ、投与された抗菌薬の影響を受けることなく、血液培養法に比べて約４倍の感度で起因菌を検出することができ、検出菌株の一致率は良好であることが明らかになっている。 そして、血液培養では、検査に少なくとも３日以上、通常は１４日程度を要するのに比較して、本発明の方法によれば全操作完了までに約８時間という極めて短時間の簡便な操作によって正確な結果を得ることができる。 従って、この方法によれば、敗血症または菌血症などの、速やかに、かつ的確な対処が必要とされる感染症の診断や予後診断のモニター等において有用マーカーが提供されるのである。
【０１１３】
また、本発明のプローブの塩基配列情報を参照してプライマーをデザインすれば、ハイブリダイゼーションを行わなくとも、ＰＣＲ法によるＤＮＡの増幅によって、感染症原因菌の同定も可能となるのである。
【０１１４】
さらに、本願発明のプローブまたはその断片をＤＮＡチップに組み込んで利用できることも明らかである。 そうすれば、ＤＮＡチップを用いて、ヘモフィリス インフルエンザ菌の存在の確定が可能となる。
【０１１５】
加えて、本発明で開示した塩基配列は、ヘモフィリス インフルエンザ菌のゲノミックＤＮＡをランダムにクローニングして得られたものであり、それ故、本発明の塩基配列の有用性はその相補鎖にまで及ぶものである。 さらに、野性株が保有するＤＮＡに変異部分が存在することは当然考えられるが、一般的に、本発明のプローブと７０％程度、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上の相同性を有する塩基配列は、本発明の目的を達成することができると考えられるので、本発明にはこれら相同配列も当然に包含されるものである。
【０１１６】
Ａ．ヘモフィリス インフルエンザ菌の検出および同定
臨床検体からのヘモフィリス インフルエンザ菌の一般的な検出および同定のための手順の具体例を、以下に詳述する。
【０１１７】
（１） 採血および血液検体の処理
臨床検体として、敗血症が疑われた患者より採取した血液検体を用いる。 各患者からヘパリン加静脈血１０ｍｌを採取し、採取した血液と血液分離試薬（塩化ナトリウム２２５ｍｇ、デキストラン（分子量：２００，０００〜３００，０００）１．５ｇ、滅菌精製水にて全量２５ｍｌに調製したもの）を４：１の割合で混和した後、３７℃で３０分間静置することにより、白血球画分（上層）を取得する。 このようにして得た白血球画分を４℃にて１６０×ｇで１０分間遠心分離することで、白血球を得る。 次に、得られた白血球のペレットに滅菌精製水１ｍｌを加えて懸濁し、これに直ちに過剰量のＰＢＳ（塩化ナトリウム１８．２４ｇ、リン酸一水素ナトリウム１２水和物６．０１２ｇ、リン酸二水素ナトリウム２水和物１．１２３ｇ、滅菌精製水にて全量１２０ｍｌにしたもの（ＰＢＳ原液）を滅菌精製水にて２０倍に希釈したもの）を加えて等張化した後に、再度４℃下で、１６０×ｇで１０分間遠心分離を行う。
【０１１８】
（２） 白血球の固定
３−アミノプロピルトリエトキシシラン（ＡＰＳ、ＳＩＧＭＡ社）をスライドグラス（商品番号ＭＳ３１１ＢＬ、日本エアーブラウン社製）にコートしたＡＰＳコートスライドグラスを使用する。
【０１１９】
ＡＰＳコートスライドグラスの作製に当たって、まず、スライドホルダーにスライドグラス（ＭＳ３１１ＢＬ）を固定した後、希釈した中性洗剤中に３０分以上浸して洗浄し、水道水で洗剤を十分に取り除き、次に、スライドグラスを精製水にて洗浄し、高温（１００℃以上）で十分に乾燥させた後、室温で放置冷却する。 その後、スライドグラスを２％ＡＰＳ含有アセトンに１分間浸し、直ちにアセトン及び滅菌精製水で順次軽く洗浄した後、風乾する。 さらに再度、スライドグラスを２％ＡＰＳ含有アセトンに１分間浸し、直ちにアセトンおよび滅菌精製水で順次軽く洗浄する。 その後、風乾する操作を行った後に、４２℃で乾燥させて、ＡＰＳコートスライドグラスを作製する。
【０１２０】
白血球画分を、４℃にて１６０×ｇで１０分間遠心分離することにより得た白血球ペレットに、少量のＰＢＳを加えて懸濁し、血球計算盤を用いて白血球数を計測する。
【０１２１】
細胞数が１×１０^５個／ウェルとなるようにＰＢＳで調製した白血球懸濁液５μｌを、ＡＰＳコートスライドグラスの各ウェルに白血球が単層に広がるように塗抹し、完全に風乾することにより、白血球をＡＰＳコートスライドグラスに支持させる。
【０１２２】
その後、カルノア固定液（エタノール：クロロホルム：酢酸＝６：３：１で混合して得た固定液）に２０分間浸した後、７５％エタノール液に５分間浸し、完全に風乾させる。
【０１２３】
（３） 白血球細胞膜の透過性亢進処理
ＰＢＳに１０分間浸した後、酵素前処理試薬（サポニン１．２５ｇ、ｔ−オクチルフェノキシポリエトキシエタノール（比重１．０６８〜１．０７５（２０／４℃）、ｐＨ（５ｗ／ｖ％） ５．５〜７．５）１．２５ｍｌ、ＰＢＳ原液２５ｍｌを混合し、滅菌精製水で全量を５０ｍｌに調製したもの）を滅菌精製水で１０倍に希釈した溶液に浸し、振とう機で１０分間振とうさせる。
【０１２４】
（４） 菌体壁の溶菌酵素処理
スライドグラス１枚につき酵素試薬（ザイモラーゼ２００単位／ｍｌ、Ｎ−アセチルムラミダーゼ１，０００単位／ｍｌ、リゾチーム１００，０００単位／ｍｌおよび／またはリゾスタフィン１００単位／ｍｌ）に酵素試薬溶解液（ＰＢＳで０．１ｍｏｌ／ｌ フェニルメチルスルフォニルフルオライド（ＰＭＳＦ）含有ジメチルスルフォキシド（ＤＭＳＯ）を１００倍希釈して調製したもの）を１ｍｌ加えて酵素試液を調製する。 その後、感染症原因菌のＤＮＡを得るために、３７℃〜４２℃の湿潤箱中で、この酵素試液１ｍｌを白血球塗抹部位に滴下し、３０分間静置することによって、感染症原因菌のＤＮＡを露出させる。 その後、０．２ｍｏｌ／ｌ 塩酸含有ＰＢＳ（ＰＢＳ原液に塩酸を加え、滅菌精製水にて２０倍希釈し、塩酸の終濃度を０．２ｍｏｌ／ｌ に調製したもの）に浸し、そのまま振とう機上で１０分間振とうする。
【０１２５】
（５） 細胞膜タンパク質のアセチル化
アセチル化試薬（トリエタノールアミン７．４６ｇと適量の塩酸を含み、適量の滅菌精製水で全量を５０ｍｌとしたもの）に無水酢酸を加え、滅菌精製水で１０倍希釈し、無水酢酸の終濃度を０．８％に調整したアセチレーション試薬にスライドグラスを浸し、振とう機上で１０分間振とうする。 その後、７５％、８５％、９８％エタノールに、順次３分間ずつ浸し、完全に風乾させる。
【０１２６】
（６） 菌体 ＤＮＡ のアルカリ処理［二本鎖 ＤＮＡ を一本鎖 ＤＮＡ に変性］
スライドグラスを、７０ｍｍｏｌ／ｌ 水酸化ナトリウム含有ＰＢＳ（ＰＢＳ原液に水酸化ナトリウムを加え、滅菌精製水で２０倍希釈し、水酸化ナトリウムの終濃度を７０ｍｍｏｌ／ｌ に調製したもの）に３分間浸す。 その後、７５％、８５％、９８％エタノールに、順次、３分間ずつ浸し、完全に風乾させる。
【０１２７】
（７） ハイブリダイゼーション
プローブ希釈液（０．５％ＳＤＳ、サケ精子ＤＮＡ６００μｌ、１００×デンハート溶液５０μｌ、ハイブリダイゼーション原液５００μｌ、ホルムアミド２２５０μｌ、５０％硫酸デキストラン１０００μｌが含まれる）にて調製したジゴキシゲニン標識ＤＮＡプローブ１５ｎｇを含有する液（プローブ液、１．０ｎｇ／μｌ）を塗抹部位に塗布し、３７℃〜４２℃の湿潤箱内に２時間静置させる。 ジゴキシゲニン標識ＤＮＡプローブは、ニックトランスレーション法で調製する。 その後、ハイブリダイゼーション洗浄液（ハイブリダイゼーション原液（塩化ナトリウム１３．１５ｇ、クエン酸三ナトリウム２水和物６．６１５ｇ、滅菌精製水にて全量７５ｍｌに調製したもの、前出）をハイブリダイゼーション原液：滅菌精製水：ホルムアミド＝５：４５：５０の割合で混合して調製したもの）を３つの染色ビンに用意し、順次４２℃で１０分間ずつ浸す。 その後、ＰＢＳに浸し、そのまま振とう機上で１０分間振とうする。
【０１２８】
プローブＨＩ−２１−８、ＨＩ−５７−３４、ＨＩ−６３−４３およびＨＩ−６５−３９に、ジゴキシゲニンを標識付けするために、ニックトランスレーション法を用いる。
【０１２９】
（８） ブロッキング
Ｉｎ ｓｉｔｕハイブリダイゼーションを行った後、ブロッキングの操作を行う。
【０１３０】
具体的には、湿潤箱内にてスライドグラス１枚につきブロッキング試薬（ウサギ正常血清２ｍｌとＰＢＳ原液０．５ｍｌを含み、滅菌精製水で全量を１０ｍｌに調製したもの）１ｍｌを塗抹部位に滴下し、これを３０分間静置する。 その後、ブロッキング試薬を除去する。
【０１３１】
（９） 標識抗体との反応
標識抗体（アルカリフォスファターゼ標識抗ジゴキシゲニン抗体溶液１．０５単位、バッファーＡ（トリエタノールアミン７４６ｍｇ、塩化ナトリウム１７．５ｍｇ、塩化マグネシウム６水和物２０．３ｍｇ、塩化亜鉛１．３６ｍｇ、ウシ血清アルブミン１０００ｍｇおよび塩酸適量を含み、滅菌精製水適量で全量を１００ｍｌに調製したもの）１２．６μｌにて全量を１４μｌに調製したもの）を、標識抗体希釈液（トリス−（ヒドロキシメチル）−アミノメタン８．４８ｍｇ、塩化ナトリウム６．１４ｍｇおよび適量の塩酸を含み、適量の滅菌精製水で全量を０．７ｍｌに調製したもの）で５０倍希釈した標識抗体液を調製し、この標識抗体液を塗抹部位に１０μｌずつ滴下し、これを３０分間静置する。 その後、標識抗体洗浄液（１ｍｌのポリソルベート２０と５０ｍｌのＰＢＳ原液を含み、滅菌精製水で全量を１００ｍｌに調製したもの）を１０倍に希釈した溶液に浸し、そのまま振とう機上で１０分間振とうさせた。 この操作を２回繰り返した後、発色前処理液−１（トリス−（ヒドロキシメチル）−アミノメタン６．０６ｇ、塩化ナトリウム２．９２ｇおよび適量の塩酸を含み、適量の滅菌精製水で全量を５０ｍｌに調製したもの）と発色前処理液−２（塩化マグネシウム６水和物５．０８ｇを含み、滅菌精製水で全量を５０ｍｌに調製したもの）を等量混合し、滅菌精製水で５倍に希釈した発色前処理液に浸し、そのまま振とう機上で１０分間振とうする。
【０１３２】
（１０） シグナル検出
スライドグラス１枚につき発色試薬［ニトロブルーテトラゾリウム（ＮＢＴ）／５−ブロモ−４−クロロ−３−インドリルフォスフェイト（ＢＣＩＰ）溶液、ｐＨ ９．０〜１０．０：ＮＢＴ ３．３ｍｇ、ＢＣＩＰ １．６５ｍｇ、Ｎ，Ｎ−ジメチルホルムアミド９９μｇ、トリス（ヒドロキシメチル）アミノメタン１２１ｍｇ、適量の塩酸、塩化ナトリウム５８．４ｍｇおよび塩化マグネシウム６水和物１０１．６ｍｇを含み、適量の滅菌精製水で全量を１０ｍｌに調製したもの］１ｍｌを、０．２μｍシリンジトップフィルターを装着したディスポーザブルシリンジを用いてろ過しながら、スライドグラスの塗抹部位に滴下し、湿潤箱内で、３７℃で、３０分間遮光静置する。 その後、発色試薬洗浄液（トリス−（ヒドロキシメチル）−アミノメタン６０６ｍｇ、エチレンジアミン四酢酸二ナトリウム２水和物１８６ｍｇおよび適量の塩酸を含み、適量の滅菌精製水で全量を５０ｍｌに調製したもの）を１０倍に希釈した溶液に５分間浸し、風乾した後、対比染色液（ファストグリーンＦＣＦ（食用緑色３号）５０ｍｇ、滅菌精製水にて全量５０ｍｌに調製したもの）を１０倍に希釈した溶液および１％酢酸溶液に浸す。 その後、前記発色試薬洗浄液を１０倍に希釈した溶液に再度浸して余分の対比染色液を洗い流し、完全に風乾させる。
【０１３３】
（１１） 判 定
判定は、光学顕微鏡で鏡検（×１，０００）した場合に、単一のウェル内の対比染色液により染まった細胞に於いて、青紫色の発色シグナルが１つでも認められた場合に「陽性」と判定する。
【０１３４】
Ｂ．貪食サンプルからのヘモフィリス インフルエンザ菌の検出
貪食サンプルからのヘモフィリス インフルエンザ菌の一般的な検出手順の事例を以下に詳述する。
【０１３５】
（１） 貪食サンプルの作製
（ｉ） Ｕ９３７細胞の調製
３７℃、５％炭酸ガスインキュベーター内で、ＲＰＭＩ １６４０培地（２５ｍｌ）を入れた細胞培養フラスコ（１７５ｃｍ^３）内にて、Ｕ９３７細胞（ヒト単球株化細胞、ＡＴＣＣ ＣＲＬ−１５９３．２）を培養する。 次に、Ｕ９３７細胞培養液を、５０ｍｌの遠沈管に入れ、４℃、２２０×ｇで１０分間遠心分離し、Ｕ９３７細胞を回収する。 その後、回収したＵ９３７細胞を、２００μｌのＰＢＳで懸濁し、血球計算盤で細胞数を計算し、細胞数を約１×１０^４個／μｌ〜約２×１０^４個／μｌに調製する。
【０１３６】
（ｉｉ） 細菌貪食サンプルの調製
ヘモフィリス インフルエンザ菌を、５ｍｌのＢＨＩ培養液に植菌し、３７℃で８時間以上培養する。 培養した菌液を、４℃、２，０００×ｇで１０分間遠心分離して集菌する。 上清を捨てた後、菌のペレットを５ｍｌのＰＢＳで懸濁し、再度４℃、２，０００×ｇで１０分間遠心分離して集菌する。 集菌した菌を５ｍｌのＰＢＳで懸濁した後、ＰＢＳにて希釈して、吸光度計により菌液の濁度（Ｏ．Ｄ．＝６００ｎｍ）を、０．０１６〜０．０２４にしたものを１５ｍｌ調製する。 このようにして得た菌液は、１７５ｃｍ^３の培養用フラスコに移し、３０分間室温で静置させる。
【０１３７】
ヘパリン加健常ヒト血液５０ｍｌを採取し、血球分離試薬を４：１の割合で加え、３７℃で３０分間静置し、白血球画分を分取し、これをＰＢＳで５０ｍｌにする。 培養用フラスコ内の上清を静かに捨て、ＰＢＳで希釈した白血球画分を１０ｍｌずつフラスコに加え、室温で１０分間静置させる。 培養用フラスコ内の上清を捨て、フラスコの底に付着した白血球を０．０２％ＥＤＴＡ含有ＰＢＳ １０ｍｌで１５ｍｌの遠沈管に回収し、４℃、１４０×ｇ〜１８０×ｇで１０分間遠心分離し、白血球を収集する。 収集した白血球に赤血球の混入が認められたときは、１ｍｌの滅菌精製水にて白血球の沈渣を穏やかに懸濁して溶血させた後、１４ｍｌのＰＢＳを加えて等張化し、再度４℃、１４０×ｇ〜１８０×ｇで１０分間遠心分離を行い、白血球を収集する。 収集した白血球をＰＢＳで懸濁し、血球計算盤にて細胞数を計測し、１×１０^４個／μｌ〜５×１０^４個／μｌに調製する。
【０１３８】
このようにして得られた貪食サンプルを、ＨＩ貪食サンプルとする。
【０１３９】
当該貪食サンプルの利点として、（１） ヘモフィリス インフルエンザ菌が検出される臨床検体の入手が困難であり、そのため本発明のプローブの特異性評価などの各種試験ができないという問題点を、臨床検体の代わりに人為的に作製した当該貪食サンプルを使用することにより回避できること、（２） 本発明のプローブまたはキット等の特異性試験、感度試験、再現性試験などの性能試験に利用できること、（３） 貪食サンプルで得られた各種試験結果を、臨床検体に適用できること、（４） 温度条件、反応時間条件、酵素処理条件などの各種条件設定に利用できること、などが挙げられる。
【０１４０】
（ｉｉｉ） 塗抹固定
（ｉ）で得たＵ９３７細胞と、（ｉｉ）で調製したＨＩ貪食サンプルを、ＡＰＳコートスライドグラスの各ウェルに５μｌずつ塗抹し、風乾させる。
【０１４１】
次に、カルノア固定液にスライドグラスを２０分間浸した後、７５％エタノールに５分間浸し、カルノア固定液を洗浄して風乾させた後、試験に使用するまで４℃で保存する（Ａ（２）の項目を参照）。 次いで、固定サンプルの前処理を、Ａ（３）の項目に記載の手順に従って行う。
【０１４２】
（２） 貪食サンプルの規格及び試験方法
（ｉ） 細胞数
細菌貪食サンプルのスライドグラスに塗抹固定する細胞数を、約５．０×１０^４〜約２．５×１０^５個／ウェルとし、また、Ｕ９３７細胞の細胞数を、約５．０×１０^４〜約１．０×１０^５個／ウェルとする。
【０１４３】
（ｉｉ） 貪食率
スライドグラスに塗抹固定した細菌貪食サンプルをアクリジンオレンジ染色液で染色し、蛍光顕微鏡（×１，０００）で無作為に約２００個の細胞を計測する。
【０１４４】
計測した細胞の中で、細胞内に細菌を貪食している細胞（貪食に特徴的な形態変化が認められた細胞）を陽性細胞とし、以下の数式に従って、貪食率を算出する。
【０１４５】
【数１】

【０１４６】
（３） 試験方法
調製した貪食サンプルを検体とする。 使用したＨＩ貪食サンプルを塗抹したスライドグラスを用いて、各プローブの特異性を検討する。
【０１４７】
（４） 結 果
プローブＨＩ−２１−８、ＨＩ−５７−３４、ＨＩ−６３−４３およびＨＩ−６５−３９とともに、貪食細胞中に取り込まれたヘモフィリス インフルエンザ菌由来のＤＮＡと特異的にハイブリダイズすることが判明する。 また、（プローブＨＩ−２１−８、ＨＩ−５７−３４、ＨＩ−６３−４３およびＨＩ−６５−３９の二つ以上を含む）混合プローブによれば、単独に比較して強いシグナルが得られることが判明する。 このことから、使用するプローブは複数種混合して使用するのが好ましい。
【０１４８】
貪食サンプルで得られた結果は、臨床検体に適用できるので、本発明のプローブは臨床検体においても有用であることが証明される。
【０１４９】
【実施例】
本発明を、実施例に沿って以下に詳細かつ具体的に説明するが、これら実施例の開示に基づいて、本発明が限定的に解釈されるべきでないことは勿論である。
【０１５０】
実施例１：ヘモフィリス インフルエンザ菌検出用プローブの調製
臨床菌株ヘモフィリス インフルエンザ菌を、チョコレートＩＩ寒天培地（日本ベクトンディッキンソン社製）で一晩培養し、培養菌体を集菌して、溶菌工程においてＮ−アセチルムラミダーゼＳＧを加えた。 そして、Ｓａｉｔｏ−Ｍｉｕｒａ変法（”Ｐｒｅｐａｒａｔｉｏｎ ｏｆ ｔｒａｎｓｆｏｒｍｉｎｇ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｉｃ ａｃｉｄ ｂｙ ｐｈｅｎｏｌｔｒｅａｔｍｅｎｔ”，Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ａｃｔａ ｖｏｌ．７２，ｐｐ．６１９−６２９（１９６３））に準じて、ゲノミックＤＮＡを抽出した。
【０１５１】
抽出したＤＮＡ １０μｇを制限酵素ＨｉｎｄＩＩＩ １０Ｕで完全消化し、ベクターｐＧＥＭ−３Ｚ（ＰＲＯＭＥＧＡ社製）にランダムクローニングした。 クローン化されたプラスミドＤＮＡを後出の実施例３−（１）に記載の方法に従って抽出し、ナイロンフィルターに転写した。 ヘモフィリス インフルエンザ菌標準菌株ゲノミックＤＮＡをジゴキシゲニン−１１−ｄＵＴＰでラベルしたものをプローブに用い、サザンブロット ハイブリダイゼーションを実施した。 すなわち、ナイロンフィルターを４５％ホルムアミド、５×ＳＳＣ、４２℃の条件下で終夜ハイブリダイゼーションを行った後、実施例２に記載の方法に従い、ナイロンフィルターの洗浄、発色を実施した。 その結果、ヘモフィリス インフルエンザ菌ゲノミックＤＮＡと特異的に反応した４種のＤＮＡ断片が選抜された。
【０１５２】
なお、ここで選抜された各プローブを、プローブＨＩ−２１−８、ＨＩ−５７−３４、ＨＩ−６３−４３およびＨＩ−６５−３９と命名した。
【０１５３】
実施例２：プローブの種特異性の検討
実施例１で選抜した各プローブと各種感染症原因菌株のＤＮＡとの反応性を、以下の方法により検討した。
【０１５４】
まず、検討対象菌株として、ヘモフィリス インフルエンザ（Ｈａｅｍｏｐｈｉｌｕｓ ｉｎｆｌｕｅｎｚａｅ 臨床分離株）、ヘモフィリス パラインフルエンザ（Ｈａｅｍｏｐｈｉｌｕｓ ｐａｒａｉｎｆｌｕｅｎｚａｅ 臨床分離株）、スタフィロコッカス アウレウス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ ＡＴＣＣ１２６００）、スタフィロコッカス エピデルミディス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｅｐｉｄｅｒｍｉｄｉｓ ＡＴＣＣ１４９９０）、エシェリヒア コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＡＴＣＣ１１７７５）、クレブシエラ ニューモニエ（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ 臨床分離株）、エンテロバクター クロアカエ（Ｅｎｔｅｒｏｂａｃｔｅｒ ｃｌｏａｃａｅ 臨床分離株）、エンテロコッカスフェカーリス（Ｅｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓ ＡＴＣＣ１９４３３）、シトロバクター フレウンディ（Ｃｉｔｒｏｂａｃｔｅｒ ｆｒｅｕｎｄｉｉ 臨床分離株）、バクテロイデス フラジリス（Ｂａｃｔｅｒｏｉｄｅｓ ｆｒａｇｉｌｉｓ ＳＭＵＭ−２２７５）、シュードモナス ジミヌタ（Ｐｓｅｕｄｏｍｏｎａｓ ｄｉｍｉｎｕｔａ ＩＦＯ１４２１３）、ミクロコッカス ルテウス（Ｍｉｃｒｏｃｏｃｃｕｓ ｌｕｔｅｕｓ ＪＣＭ１４６４）およびＵ９３７（ヒトゲノムＤＮＡ）を準備した。
【０１５５】
そして、各臨床菌株に関して、実施例１に記載の方法に従って、各菌株が保有するＤＮＡを抽出し、抽出したＤＮＡの一定量（例えば、１０〜１００ｎｇ）をナイロンフィルターにスポットして、アルカリ変性したものをドット ブロット ハイブリダイゼーションの試料とした。 次いで、Ｄｉｇｏｘｉｇｅｎｉｎ−１１−ｄＵＴＰ（ＢＲＬ社製）でラベルしたプローブで、マニアティスのマニュアル（Ｔ． Ｍａｎｉａｔｉｓ， ｅｔ ａｌ．， ＭｏｌｅｃｕｌａｒＣｌｏｎｉｎｇ（Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ Ｓｅｃｏｎｄ Ｅｄｉｔｉｏｎ）， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｕｒ Ｌａｂｏｒａｔｏｒｙ （１９８９））に従い、４５％ホルムアミド、５×ＳＳＣ、４２℃の条件下で、終夜ハイブリダイゼーションを実施した。 終夜ハイブリダイゼーションを終えた試料に関して、マニュアルに従い、５５℃にて０．１×ＳＳＣ、０．１％ＳＤＳによる２０分間の洗浄を２回行った後に、Ａｎｔｉ−Ｄｉｇ−ＡＬＰ ｃｏｎｊｕｇａｔｅｓ（ＢＲＬ社製）で検出および発色させ、ハイブリダイゼーションの状況を確認した。
【０１５６】
その結果、プローブＨＩ−２１−８、ＨＩ−５７−３４、ＨＩ−６３−４３およびＨＩ−６５−３９は、いずれもヘモフィリス インフルエンザ菌のゲノムＤＮＡに対してのみ特異的に反応することが確認された。
【０１５７】
実施例３：塩基配列の解析
実施例２で種特異性が確認されたＤＮＡプローブ（計４本）の塩基配列を、下記の方法に従って決定した。
【０１５８】
（１） プラスミド ＤＮＡ の調製
サブクローン化された（塩基配列を決定すべき）挿入断片をｐＧＥＭ−３Ｚ（Ｐｒｏｍｅｇａ）に含んだ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ Ｋ−１２，ＪＭ１０９形質転換体を、５ｍｌのＬｕｒｉａ−Ｂａｃｔａｎｉ Ｍｅｄｉｕｍ（ｂａｃｔｏ−ｔｒｙｐｔｏｎｅ，１０ｇ／ｌ；ｂａｃｔｏ−ｙｅａｓｔ ｅｘｔｒａｃｔ、５ｇ／ｌ；ＮａＣｌ，１０ｇ／ｌ；５Ｎ ＮａＯＨでｐＨ ７．０に調整）に植菌し、一晩培養した。 培養液を遠心分離（５，０００ｒｐｍ、５分間）して集菌した。 沈殿物に２．５ｍｇ／ｍｌの濃度でリゾチーム（Ｓｉｇｍａ）を含む５０ｍＭグルコース／５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ ８．０）／１０ｍＭ ＥＤＴＡ溶液を１００μｌ 加え、室温で５分間放置した。 得られた懸濁液に１％の濃度でドデシル硫酸ナトリウム（Ｓｉｇｍａ）を含む０．２Ｍ水酸化ナトリウム水溶液を加えて混合した。
【０１５９】
５Ｍ酢酸カリウム水溶液（ｐＨ ４．８）１５０μｌ をさらに加えて混合し、１５分間氷冷した。 そして、遠心分離（１５，０００ｒｐｍ、１５分間）して得た上清を、フェノール／ＣＨＣｌ_３処理し、上清に２倍量のエタノールを加え、さらに遠心分離（１２，０００ｒｐｍ、５分間）して沈澱を得た。 この沈澱物を１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ ７．５）／０．１ｍＭ ＥＤＴＡ溶液１００μｌ に溶解し、１０ｍｇ／ｍｌ ＲＮａｓｅＡ（Ｓｉｇｍａ）溶液を加え、室温で１５分間放置した。 この調製物に０．１Ｍ酢酸ナトリウム水溶液（ｐＨ ４．８）を３００μｌ加え、フェノール／ＣＨＣｌ_３処理し、上清にエタノールを加えて沈殿を得た。 この沈澱物を乾燥し、１０μｌの蒸留水に溶解したものをＤＮＡ試料とした。
【０１６０】
（２） 塩基配列決定の前処理
塩基配列決定の前処理をＡｕｔｏＲｅａｄ（登録商標）Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（ファルマシア製）を用いて行った。
【０１６１】
まず、鋳型となるＤＮＡが３２μｌ 溶液中に５〜１０μｇの濃度になるように調整した。 １．５ｍｌのミニチューブ（エッペンドルフ）に、鋳型ＤＮＡ３２μｌを移し、２Ｍ水酸化ナトリウム水溶液を８μｌ 加えて穏やかに混合した。 そして、軽く遠心した後、室温で１０分間放置した。 ３Ｍ酢酸ナトリウム（ｐＨ ４．８）７μｌ と蒸留水４μｌ を加え、さらにエタノールを１２０μｌ 加えて混合し、エタノール−ドライアイス上で１５分間放置した。 そして、１５分間遠心分離して沈澱したＤＮＡを集め、注意しながら上清を除去した。 得られた沈殿物を７０％エタノールで洗浄し、１０分間遠心分離した。 そして、注意しながら再度上清を除去し、減圧条件下で沈澱物を乾燥した。
【０１６２】
沈殿物を蒸留水１０μｌに溶解し、蛍光性のプライマー〔Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｉｍｅｒ，Ｕｎｉｖｅｒｓａｌ Ｐｒｉｍｅｒ；５’−Ｆｌｕｏｒｅｓｃｅｉｎ−ｄ［ＣＧＡＣＧＴＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ（配列番号：５）］−３’、（１．６ｐｍｏｌ／μｌ ；０．４２Ａ_２６０ｕｎｉｔ／ｍｌ）；Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒ，５’−Ｆｌｕｏｒｅｓｃｅｉｎ−ｄ［ＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣ（配列番号：６）］−３’ （２．１ｐｍｏｌ／μｌ；０．４２Ａ_２６０ｕｎｉｔ／ｍｌ）〕２μｌ（０．４２Ａ_２６０ｕｎｉｔ／ｍｌ、４〜６ｐｍｏｌ）とアニーリング用緩衝液２μｌを加え穏やかに混合した。 そして、軽く遠心した後、６５℃で５分間熱処理を行い、素早く３７℃の条件下に置き、そこで１０分間保温した。 保温後１０分以上室温で放置し、軽く遠心した。 そして、延長用緩衝液１μｌとジメチルスルホキシド３μｌを加えたものを試料とした。
【０１６３】
４本のミニチューブにＡ、Ｃ、ＧおよびＴと記入し、それぞれのチューブにＡＭｉｘ（ｄｄＡＴＰをｄＡＴＰ、ｄＣＴＰ、ｃ７ｄＧＴＰおよびｄＴＴＰと共に溶解したもの）、Ｃ Ｍｉｘ（ｄｄＣＴＰをｄＡＴＰ、ｄＣＴＰ、ｃ７ｄＧＴＰおよびｄＴＴＰと共に溶解したもの）、Ｇ Ｍｉｘ（ｄｄＧＴＰをｄＡＴＰ、ｄＣＴＰ、ｃ７ｄＧＴＰおよびｄＴＴＰと共に溶解したもの）およびＴ Ｍｉｘ（ｄｄＴＴＰをｄＡＴＰ、ｄＣＴＰ、ｃ７ｄＧＴＰおよびｄＴＴＰと共に溶解したもの）を、２．５μｌ ずつ分注した。
【０１６４】
なお、それぞれの溶液は使用時までは水中で保存し、使用時には３７℃で１分間以上保温してから使用した。 希釈したＴ７ ＤＮＡポリメラーゼ（Ｐｈａｒｍａｃｉａ；６〜８ｕｎｉｔｓ／２μｌ）２μｌをＤＮＡ試料に加え、ピペッティングもしくは穏やかな混合により、完全に混合した。 混合後すぐに、この混合液を４．５μｌずつ保温しておいた４種の溶液に分注した。 なお、分注に際しては新しいチップを用いた。
【０１６５】
３７℃で５分間保温し、停止溶液を５μｌずつそれぞれの反応液に加えた。 この分注においても、新しいチップを用いた。 ９０℃で２〜３分間保温し、すぐに氷中で冷却した。
【０１６６】
そして、電気泳動には、１レーン当たり４〜６μｌの試料を泳動した。
【０１６７】
（３） 塩基配列の決定
実施例１〜２に開示した、ヘモフィリス インフルエンザ菌が保有するＤＮＡに対して特異性を有するプローブそれぞれの塩基配列の決定を、泳動温度４５℃、泳動時間６時間として、Ａ．Ｌ．Ｆ． ＤＮＡ Ｓｅｑｕｅｎｃｅｒシステム（ファルマシア社製）を用いて行った。 各上流と下流から明らかになった配列から順次プライマーをデザインし、上記の操作を繰り返した。
【０１６８】
その結果、プローブＨＩ−２１−８（配列番号１）、ＨＩ−５７−３４（配列番号２）、ＨＩ−６３−４３（配列番号３）およびＨＩ−６５−３９（配列番号４）の各塩基配列の全容が明らかになった。
【０１６９】
【発明の効果】
このように、本発明の検出用プローブによれば、それをｉｎ ｓｉｔｕハイブリダイゼーションに適用することで、極めて短時間の内に、検出対象菌に対して安定なシグナルを発現することができるため、迅速かつ的確な検査結果をもたらすことが可能となる。
【０１７０】
また、これにより、ヘモフィリス インフルエンザ菌のみならず、敗血症や菌血症などの疾患に対する診断材料を医療現場に迅速に提供でき、人命救助の観点からも多大な貢献が期待されるものである。
【０１７１】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention generally relates to improvements in the detection technology of Haemophilus influenzae, and in particular, novel probes for the detection and identification of Haemophilus influenzae, and the detection and identification of Haemophilus influenzae using these probes. Method and related technology.
[0002]
2. Description of the Related Art
Haemophilus influenzae is a Gram-negative bacillus that inhabits the epithelial cells of the upper respiratory tract. Haemophilus influenzae is roughly classified into a strain having a capsule and a strain having no capsule. Among these, the capsulated strains are classified into six types a to f according to the serotype of the capsular polysaccharide. On the other hand, strains without a capsule are classified as non-typeable. Strains having a capsule are less frequently isolated from clinical specimens, but are generally highly pathogenic, and among them, serotype b is known as the most pathogenic bacteria. In particular, type b causes severe pathological conditions, mainly in children and infants, such as acute purulent meningitis, acute epiglottitis, pneumonia, cellulitis, sepsis, and arthritis. A quick and accurate method of diagnosis and detection has been desired. Also, due to the shortage of pediatricians, doctors who do not specialize in pediatrics are keen to find a method that can easily diagnose and detect Haemophilus influenzae.
[0003]
In cases of acute bronchitis, pneumonia, pharyngitis, chronic bronchitis, chronic respiratory tract infections, otitis media, and sinusitis in adults in Japan, non-typeable Haemophilus influenzae bacteria often cause pathogens. In particular, severe pneumonia often occurs in patients with bronchiectasis accompanied by chronic bronchitis, elderly people, and the like, and establishment of an early diagnosis method is required. However, clinical symptoms are the same as other bacterial pneumonia such as fever, chills, dyspnea, cough, purulent sputum, chest pain, etc., so that a definitive diagnosis cannot be made only by clinical symptoms. In addition, since Haemophilus influenzae is a resident of the pharynx, there is a problem that even if Haemophilus influenzae is detected from the upper respiratory tract by culturing, it does not necessarily lead to abnormal findings.
[0004]
Currently, diagnosis of respiratory tract infections caused by Haemophilus influenzae is based on clinical symptoms, blood tests (white blood cell increase based on neutrophils, C-reactive protein (CRP) positive, increased erythrocyte sedimentation, etc.), diagnostic imaging, gram staining of sputum It is performed comprehensively according to the results of culture and the like.
[0005]
Diagnosis of meningitis due to Haemophilus influenzae is based on clinical symptoms of purulent meningitis such as increased cerebrospinal fluid pressure, increased number of cells in cerebrospinal fluid, protein positivity, low glucose, and gram staining and culture of cerebrospinal fluid sediment. This is done based on the results. Furthermore, epiglottitis is diagnosed by clinical symptoms, laryngoscopic findings, Gram stain of epiglottis punctures obtained by endotracheal intubation and culture results. In the case of sepsis, the diagnosis is made by blood culture, and in the case of arthritis, the diagnosis is made by staining and culture of a joint puncture solution.
[0006]
As described above, the diagnosis method differs depending on the disease state even if Haemophilus influenzae is involved.Furthermore, a wide variety of tests are required, so that a prompt definitive diagnosis cannot be made and the diagnosis is expensive. was there. Furthermore, in addition to having a long culture time for the bacterium, a further 3 to 4 days of cultivation are required before a drug sensitivity result is obtained, which makes rapid diagnosis difficult.
[0007]
In the treatment of infectious diseases, it is theoretically important to first determine the causative bacterium and, secondly, to carry out appropriate treatment according to the causative bacterium. In order to achieve this, it is necessary to perform a bacterial test or the like in advance, but it may be urgently required in clinical practice, and in reality, these treatments are hardly implemented. In other words, many physicians make diagnoses and predict pathogenic bacteria based on their own treatment experience, administer antibacterial drugs against the predicted pathogenic bacteria, observe the course of treatment, and terminate treatment or identify the pathogenic bacteria. Waiting for the test results and taking other measures such as changing antibiotics. According to these current treatment strategies, there is a high possibility that antibiotics that are ineffective against the causative agent will be administered, and patients may be left at risk without appropriate treatment. There is no doubt that there is an increase in medical expenses due to wasteful use of antibacterial drugs. In addition, if antibiotics are administered in large quantities at the time of suspected infection, bacterial enrichment and growth may be suppressed even if the specimen contains causative bacteria. In fact, the possibility of culturing causative bacteria from these specimens is extremely low.
[0008]
As a conventional method for detecting bacteria including H. influenzae, there is a detection method using ampicillin-resistant H. influenzae (see, for example, Patent Document 1). However, there is a lack of a specific method for handling clinical samples, and There is no proof of cross-reaction with other bacteria mixed in the specimen.
[0009]
Also, a method for preparing a polyclonal antibody against the outer membrane protein of Haemophilus influenzae and a diagnostic method using the antibody have been proposed (for example, see Patent Document 2).
[0010]
Furthermore, a method of detecting various microorganisms using an antibody against ribosomal proteins has been devised (see, for example, Patent Document 3). However, the measurement result by such an immunological test method is the test result by a culture method. In many cases, false positive or false negative results are shown, and the procedure remains complicated.
[0011]
As known detection kits related to influenzae, RapID NH kit (Amco), Slidex meningi kit (bioMerieu), influenzae gene detection reagent (Wasunaga Pharmaceutical) and the like are commercially available. However, they have a problem that they are not specific to Haemophilus influenzae, and a problem that the detection rate decreases when a clinical specimen is used.
[0012]
In order to solve such problems, the present applicant has invented a method for detecting and / or identifying a foreign microorganism phagocytosed by phagocytic cells (see Patent Document 4). That is, according to this method, genes derived from foreign microorganisms present in phagocytic cells are detected by in situ hybridization using a probe capable of specifically hybridizing to these genes. Specifically, this method involves fixing phagocytes obtained from a biological specimen derived from a living body, subjecting these phagocytes to a treatment to enhance the permeability of the cell membrane, and infecting the phagocytes into the phagocytes. Exposing the DNA of the causative bacterium, performing in situ hybridization using a DNA probe for detection capable of hybridizing to the DNA of the causative bacterium under stringent conditions, and determining the presence or absence of a hybridization signal Detecting and / or identifying infectious disease-causing bacteria.
[0013]
When blood of a patient suspected of sepsis was tested according to this method, bacteria were detected with about four times the sensitivity as compared with the blood culture method, and the determination could be completed within 24 hours. This method has been spotlighted in the field of infectious diseases.
[0014]
[Patent Document 1]
JP-A-2000-342268
[Patent Document 2]
Tokiko 06-64065
[Patent Document 3]
International Publication Pamphlet No. WO00 / 06603
[Patent Document 4]
Tokuhei 07-40
[0015]
As described above, the present invention provides a novel probe useful for detection of Haemophilus influenzae, particularly a hybridization probe, and by using these probes, improves the detection efficiency and the detection sensitivity over the conventional detection method. The aim is also to realize an excellent detection method and detection means.
[0016]
[Hands to solve the problem]
The present invention has been made in view of the above-mentioned disadvantages recognized in the prior art, and its gist is to show a specific cross-reactivity with Haemophilus influenzae, and (a) A) a base sequence according to any of SEQ ID NOs: 1 to 4; (b) a base sequence having 70% or more homology (homology) with the base sequence (a); or (c) a base sequence (a) and And / or a probe for detecting Haemophilus influenzae comprising a base sequence complementary to (b).
[0017]
According to the present invention, there is also provided a method for detecting Haemophilus influenzae using the above-described detection probe. This detection method comprises the steps of (a) immobilizing a phagocyte derived from a living body obtained from a clinical specimen on a support, (b) performing a chemical treatment to enhance the permeability of the cell membrane of the immobilized phagocyte, and (c) (D) performing in situ hybridization between the obtained chromosomal DNA and the detection probe of the present invention under stringent conditions, and (e) ) Detecting a hybridization signal.
[0018]
Further, according to another aspect of the present invention, there is provided a method for identifying Haemophilus influenzae. This identification method comprises (1) obtaining chromosomal DNA of an infectious disease-causing bacterium of unknown species, (2) preparing a primer comprising at least a part of the nucleotide sequence constituting the detection probe of the present invention, and (3) Amplifying the DNA by polymerase chain reaction (PCR) in a coexistence system of the obtained chromosomal DNA and the primer, and (4) detecting the amplified DNA.
[0019]
According to still another aspect of the present invention, there is provided a method for observing a gene of a foreign microorganism phagocytosed by a phagocyte, particularly, a gene of Haemophilus influenzae. This observation method comprises (i) immobilizing a phagocyte derived from a living body obtained from a clinical sample on a support, (ii) performing a chemical treatment to enhance the permeability of the cell membrane of the immobilized phagocyte, and (iii) Obtaining chromosomal DNA of an infectious disease-causing bacterium contained in cells, (iv) performing in situ hybridization between the obtained chromosomal DNA and a detection probe of the present invention under stringent conditions, and (v) a hybridization signal. (Vi) repeating steps (i) to (v), and (vii) monitoring the change in the hybridization signal over time.
[0020]
In addition, according to still another aspect of the present invention, the detection probe of the present invention, a phagocyte-containing sample preparation device, a support carrier, a chemical treatment agent for cell membranes, a DNA exposure treatment agent, a hybridization tool, and hybridization signal detection A kit for detecting Haemophilus influenzae including the device is provided.
[0021]
According to still another aspect of the present invention, there is provided a DNA chip comprising a chip substrate and the detection probe of the present invention or a fragment thereof, one end of which is fixed to the surface of the substrate.
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0023]
Definition
First, the term "clinical specimen" used herein is a general term for a clinical specimen containing phagocytes derived from a living body, such as blood, tissue fluid, lymph, cerebrospinal fluid, pus, mucus, and runny nose. And bodily fluids such as sputum. In addition, depending on the disease state such as diabetes, renal disorder, liver disorder, etc., in addition to urine, ascites fluid, dialysis drainage, etc., the washing fluid after washing the nasal cavity, bronchi, skin, various organs, bones, etc., may also be derived from living organisms. Since they contain cells, they are also included in the category of clinical specimens. In addition, tissues such as skin, lung, kidney, and mucous membrane can be used as the clinical specimen of the present invention. This means that macrophages, which are one of the phagocytes, include various types of monocytes, alveolar macrophages, peritoneal macrophages, fixed macrophages, free macrophages, Hansemann macrophages, inflammatory macrophages, liver Kupffer cells, brain microglial cells, etc. Because the morphology changes, not only blood but also tissues containing them can be used as the clinical specimen of the present invention. For example, from a patient suspected of having nephritis, renal tissue is collected according to a renal biopsy, cells are detached by using an enzyme such as trypsin to obtain phagocytes present in the tissue, and the obtained phagocytes are collected. By using it, the causative microorganism of nephritis can be detected and identified.
[0024]
Next, the term "phagocytic cell" as used herein refers to a cell capable of taking up foreign substances, including foreign microorganisms, into its own cells, such as macrophages, monocytes, and neutrophils. Spheres, eosinophils and the like. In addition, phagocyte systems such as U937 cells and HL60 cells can also be suitably used in the present invention.
[0025]
The phagocyte (leukocyte) fraction can be obtained from a clinical specimen by a known method. For example, about 5 ml of heparinized venous blood (10 ml if the number of white blood cells is low) is collected, and this blood and a blood separation reagent [containing 225 mg of sodium chloride and 1.5 g of dextran (molecular weight 200,000 to 300,000); Prepared in sterile purified water to a total volume of 25 ml) at a ratio of about 4: 1, and then at about 10 ° C to about 40 ° C for about 15 minutes to about 120 minutes, preferably at about 37 ° C. After standing for about 30 minutes, the leukocyte fraction (upper layer) can be obtained.
[0026]
Phagocyte fixation
The leukocyte fraction thus obtained is treated at about 0 ° C. to about 20 ° C. at about 100 × g to about 500 × g for about 3 minutes to about 60 minutes, preferably at about 4 ° C. Leukocytes can be obtained by centrifugation at 140 × g to about 180 × g for about 10 minutes.
[0027]
When erythrocytes are mixed during centrifugation, it is preferable to perform a hemolysis operation. For example, immediately after adding and suspending 1 ml of sterile purified water to a leukocyte pellet, an excess amount of PBS [18.24 g of sodium chloride, 6.012 g of sodium monohydrogen phosphate decahydrate and 6.012 g of sodium dihydrogen phosphate) is immediately added. A solution containing 1.123 g of hydrate and adjusted to a total volume of 120 ml with sterile purified water (hereinafter, simply referred to as "PBS stock solution"), diluted 20-fold with sterile purified water, and obtained (hereinafter, referred to as "PBS stock solution"). , Simply referred to as “PBS”), and then centrifuged again at a temperature of about 4 ° C. at about 140 × g to about 180 × g for about 10 minutes.
[0028]
Alternatively, even without such centrifugation, phagocytic cells can be adhered to a slide glass in the following manner by utilizing the inherent adhesive ability. As a method for fixing leukocytes, for example, Carnoy's fixation can be performed. Specifically, a leukocyte pellet is placed on a carrier capable of supporting leukocytes (supporting carrier), and is added to a Carnoy's fixative (a solution obtained by mixing ethanol: chloroform: acetic acid = 6: 3: 1) for about 20 minutes. After soaking, soak for about 5 minutes in about 50% to about 90%, preferably about 75% ethanol solution, and air-dry completely.
[0029]
As such a support, those formed of an insoluble material are preferable. For example, glass, metal, synthetic resin (polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylate, nylon, polyacetal, fluororesin) And polysaccharides (cellulose, agarose, etc.) can be suitably used. Examples of the shape of the insoluble support carrier include various shapes such as a plate, a tray, a sphere, a fiber, a bar, a disk, a container, a cell, and a tube.
[0030]
In particular, a preferred support carrier in the embodiment of the present invention is a slide glass. As such a slide glass, for example, there is a slide glass (product number MS311BL: manufactured by Air Brown Japan). This MS311BL slide glass has 14 circular wells with a diameter of 5 mm on its surface.
[0031]
Further, in consideration of actual application, an APS-coated slide glass having 3-aminopropyltriethoxysilane (APS, SIGMA) coated on its surface for the purpose of improving the adhesiveness of cells to a support carrier. Is preferred. In addition, a slide glass coated with poly-L-lysine or gelatin can be preferably used. In the procedure for preparing these APS-coated slide glasses, first, the slide glasses are fixed to a slide holder. The fixed slide glass is washed by immersing it in a diluted neutral detergent for 30 minutes or more, thoroughly removing the detergent with tap water, washing with purified water, and then sufficiently drying at a high temperature (100 ° C. or more). Leave to cool at room temperature. Next, the slide glass is immersed in acetone containing 2% APS for 1 minute, immediately washed lightly with acetone and sterile purified water, and then air-dried. Further, the slide glass was immersed again in acetone containing 1 to 10% APS for 1 minute, immediately washed lightly with acetone and sterile purified water, air-dried, and then about 20 ° C to about 60 ° C, preferably about 42 ° C. APS-coated slide glass is obtained by drying in
[0032]
When the leukocytes are supported on the surface of the APS-coated slide glass, it is preferable that each well be smeared such that the leukocytes spread in a single layer and air-dried. The density of phagocytes to be immobilized (x cells / ml) was about 5 × 10⁶Pieces / ml <x pieces / ml <about 1 × 10⁸Pcs / ml, preferably about 1 × 10⁷Pieces / ml ≦ x pieces / ml ≦ about 5 × 10⁷Adjust to individual pieces / ml. Correspondingly, the number of white blood cells per well fixed on the APS-coated slide glass (y cells / well (5 mm in diameter)) is about 2.5 × 10 5⁴Cells / well <y cells / well <about 5 × 10⁵Cells / well, preferably about 5 × 10⁴Cells / well ≦ y cells / well ≦ about 2.5 × 10⁵Adjust to cells / well.
[0033]
Specifically, a small amount of PBS is added to a leukocyte pellet obtained by centrifuging the leukocyte fraction at about 4 ° C. at about 140 × g to about 180 × g for 10 minutes, and the leukocyte fraction is suspended. The white blood cell count is measured using a panel.
[0034]
And about 5 × 10⁴Pieces / well to about 2.5 × 10⁵5 μl of a leukocyte suspension prepared in PBS to give a cell number of cells / well was spread on each well of an APS-coated slide glass so that the leukocytes spread in a monolayer, and completely air-dried. An APS-coated slide glass fixed and supported on the surface is prepared.
[0035]
Cell membrane permeability enhancement
A treatment for enhancing the permeability of the phagocyte cell membrane is performed. As this treatment method, an APS-coated slide glass on which leukocytes are fixed and supported is immersed in PBS for about 3 to about 30 minutes, and then an enzyme pretreatment reagent (1.25 g of saponin, t-octylphenoxypolyethoxyethanol (specific gravity: 1. 068-1.075 (20/4 ° C.), pH (5 w / v%) 5.5-7.5) 1.25 ml, 25 ml of PBS stock solution were mixed and adjusted to a total volume of 50 ml with sterile purified water.) May be immersed in a solution diluted about 2- to about 50-fold with sterile purified water, and shaken with a shaker for about 3 to about 30 minutes.
[0036]
Acquisition of endogenous bacterial DNA
Next, DNA of an infectious disease-causing bacterium present in the phagocyte is obtained. Specifically, first, an enzyme reagent (N-acetylmuramidase, lysozyme and / or lysostaphin; hereinafter, simply referred to as “enzyme reagent”) per slide glass is reacted with an enzyme reagent solution (phenylmethylsulfonylfluoride). Of dimethylsulfoxide (DMSO) containing 100 μl of Ride (PMSF) diluted 100-fold with PBS] to prepare an enzyme reagent solution, followed by about 20 ° C. to about 60 ° C., preferably about 37 ° C. to about 60 ° C. In a humid box at 42 ° C., 1 ml of the enzyme reagent is dropped onto the leukocyte smear site and allowed to stand for about 10 to about 60 minutes to expose the DNA of the causative bacteria of the infectious disease. It was immersed in PBS containing hydrochloric acid (prepared by adding hydrochloric acid to a PBS stock solution and diluting 20-fold with sterile purified water to adjust the final concentration of hydrochloric acid to 0.2 mol / l). DMSO is preferably used at a concentration of less than 5% because DMSO may reduce the activity of lysozyme and lysostaphin at a concentration of 5% or more. .
[0037]
In addition to PMSF, which is a substance that retains the phagocyte morphology, other known protease inhibitors, for example, tosyl lysine chloromethyl ketone (TLCK) and mixtures thereof can also be used. In such a case, the dissolving agent such as DMSO may be appropriately changed.
[0038]
The titer range of each enzyme used as an enzyme reagent is as follows. The titer range for lysostaphin is from about 1 unit / ml to about 1,000 units / ml, preferably from about 10 units / ml to about 100 units / ml. The titer of N-acetylmuramidase ranges from about 10 units / ml to about 10,000 units / ml, preferably from about 100 units / ml to about 1,000 units / ml. And, the titer range of lysozyme is about 1,000 units / ml to about 1,000,000 units / ml, preferably, about 10,000 units / ml to about 100,000 units / ml. When the causative bacterium is a fungus such as Candida albicans, it is preferable to use zymolase alone or in combination with other enzymes, and the titer range is about 50 to about 500 units / ml, preferably , About 100 units / ml to about 500 units / ml. When zymolase is used, it is particularly preferable to use PMSF or a known protease inhibitor in combination.
[0039]
Further, the enzyme to be used can be appropriately selected depending on the difference in the cell components of the gram-positive bacteria and the gram-negative bacteria, that is, the difference in peptidoglycan or lipopolysaccharide. Particularly, regardless of the type of gram-positive bacteria and gram-negative bacteria, two or more types of enzymes can be used to more effectively lyse the cells. By using an enzyme reagent in which three types of enzymes, lysozyme, lysostaphin, and N-acetylmuramidase, are mixed, the lytic activity is increased as compared with the case where one type of enzyme is used.
[0040]
The optimal enzyme treatment temperature of the enzyme reagent is set at about 26 ° C to about 59 ° C, preferably about 37 ° C to about 42 ° C. The enzyme treatment time of the enzyme reagent is at least about 15 minutes or more, preferably about 15 minutes to about 120 minutes, or at least about 20 minutes or more, and preferably about 30 minutes to about 60 minutes.
[0041]
Regarding N-acetylmuramidase, heat-treated dried powder of Enterococcus faecalis and N-acetylmuramidase were used together with N-acetylmuramidase in a 5 mmol / l Tris-HCl buffer (pH 6.0) containing 2 mmol / l magnesium chloride. When the reaction is performed at 37 ° C. for 5 minutes, a phenomenon in which the absorbance at 600 nm decreases is observed. When heat-treated cells of Streptococcus salivarius (IFO3350) are lysed at 1 μg per minute under the conditions of 37 ° C. and pH 7.0, 1 unit is 2,000 units / mg or more. It is desirable to use N-acetylmuramidase which exhibits the activity of
[0042]
Regarding lysozyme, when reacted with Micrococcus luteus (micrococcus luteus) and lysozyme in PBS at 37 ° C. for 5 minutes, a phenomenon in which the absorbance at 600 nm decreases is observed. In addition, assuming that Micrococcus luteus has an enzyme activity of 1 unit when the absorbance at 540 nm is reduced by 0.001 per minute under the conditions of 35 ° C. and pH 6.2, an activity of 50,000 units / mg or more is obtained. It is desirable to use the indicated lysozyme.
[0043]
Regarding lysostaphin, when both Staphylococcus epidermidis and lysostaphin are reacted in PBS at 37 ° C. for 5 minutes, a phenomenon in which the absorbance at 600 nm decreases is observed. In addition, when Staphylococcus aureus is used at 37 ° C. and pH 7.5 for 10 minutes, the enzyme activity for reducing the absorbance at 620 nm from 0.240 to 0.125 in 1 minute is defined as 1 unit. It is desirable to use lysostaphin showing an activity of 500 units / mg or more.
[0044]
Zymolase (trade name: Zymolyase, Seikagaku Kogyo) is an enzyme prepared from a culture solution of Arthrobacter lutesu1 and has a high lytic activity on the cell wall of live yeast cells. An essential enzyme involved in cell wall lysis contained in zymolase is β-1,3-glucan laminaripentaohydrolase (β-1,3-glucan lanimalipentaohydrolase), which is β-1,3-linked glucose. Acts on the polymer to produce laminalipentaose as the main product. Zymolyase-100T is an enzyme purified into ammonium sulfate fraction and further purified by affinity chromatography (Kitamura, K. et al., J. Ferment. Technol., 60, 257, 1982), 100,000. It has an activity of unit / g. However, it is known that the activity of this enzyme changes depending on the type of yeast serving as a substrate, cultivation conditions and growth period (Kitamura, K. et al., J. Gen. Appl. Microbiol., 20, 323). Kitamura, K. et al., Agric. Biol. Chem., 45, 1761, 1981; Kitamura, K. et al., Agric. Biol. Chem., 46, 553, 1982). Zymolyase-100T is about 1.0 × 10 β-1,3-glucanase.⁷Unit / g, about 1.7 × 10 9 protease⁴Units / g, and about 6.0 × 10⁴Unit / g, and DNase and RNase are not found (Kitamura, K. et al., J. Gen. Appl. Microbiol., 18, 57, 1972). Further, the optimum pH of Zymolyase is about 5.5 to about 8.5, and a pH of about 6.5 to about 7.5 is particularly excellent in optimumness, and the optimum temperature is about 25 to about 55 ° C. And a temperature of about 35 to about 45 ° C. is particularly desirable.
[0045]
Furthermore, the yeast lytic spectrum (genus name) for (logarithmic growth phase cells), Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloekera, Kluyveromyces, Lipomyces, Metschkowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, Schwanniomyces and the like. In particular, the Candida genus, Candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Candida para White cis (Candida parapsilosis), Candida junk (Candida galacta), Candida Girierumonji (Candida guilliermondii), Candida krusei (Candida krusei ).
[0046]
Bacteria belonging to these genera can also be added to the application of the present invention.
[0047]
As an activator for zymolase, an SH compound such as cysteine, 2-mercaptoethanol, dithiothreitol and the like can be used. The zymolase is a reaction solution (zymolase: 0.05 to 0.1 mg / ml of a solution, 1 ml of a brewer's yeast suspension, placed at about 25 ° C. for about 2 hours using a brewer's yeast suspension as a substrate. (2 mg dry weight / ml) 3 ml, buffer: 5 ml of M / 15 phosphate buffer (pH 7.5), adjusted to a total volume of 10 ml with 1 ml of sterile purified water)₈₀₀1 unit is defined as the enzyme activity required to reduce the concentration by 30%. Zymolyase-100T has an activity of about 100,000 units / g.
[0048]
PMSF used as an enzyme reagent dissolving solution (added to protect leukocytes from proteases and maintain its form) is effective at a concentration of about 10 μmol / l or more, and has an effect of about 0.1 mmol / l or more. The concentration is preferably in the range of about 10 μmol / l to about 10 mmol / l, and more preferably in the range of about 0.1 mmol / l to about 1 mmol / l, since the deterioration of leukocyte morphology was completely suppressed. The concentration of dimethylsulfoxide (DMSO) can be used at a concentration of less than about 5%, preferably about 2% or less, and most preferably about 1%. Therefore, the enzyme reagent solution is preferably prepared by diluting 0.1 mol / l PMSF-containing DMSO with PBS by about 100 to about 1,000 times.
[0049]
After obtaining the DNA of the causative microorganism of the infectious disease, an acetylation step of the cell membrane protein may be added. Specifically, acetic anhydride is added to an acetylating reagent (containing 7.46 g of triethanolamine and an appropriate amount of hydrochloric acid, and the total volume is adjusted to 50 ml with an appropriate amount of sterilized purified water), and about 2 times with sterilized purified water. The acetylation reagent is diluted about 50-fold, preferably about 10-fold, to a final concentration of acetic anhydride of about 0.1 to about 3.0%, preferably about 0.8%. Soak the slide glass and shake on a shaker for 5-30 minutes. Thereafter, the slide glass is immersed in 75%, 85%, and 98% ethanol in order for about 2 to about 5 minutes each, and completely air-dried.
[0050]
Further, after the acetylation step, a step of alkali-treating the DNA of the causative microorganism of the infectious disease can be inserted. Thereby, the DNA of the infectious disease-causing bacteria becomes single-stranded DNA. Specifically, the slide glass was washed with PBS containing sodium hydroxide at a concentration of about 10 mmol / l to about 300 mmol / l, preferably about 70 mmol / l (sodium hydroxide was added to a PBS stock solution, and sterilized purified water was added to about 20 mmol / l). It is diluted twice and adjusted to a final concentration of sodium hydroxide of 70 mmol / l) for about 2 to about 5 minutes to carry out alkali treatment. Thereafter, the slide glass is immersed in 75%, 85%, and 98% ethanol in order for about 2 to about 5 minutes each, and completely air-dried.
[0051]
In situ Hybridization
Under stringent conditions, in situ hybridization is performed using a DNA probe for detection capable of hybridizing with DNA of the causative microorganism of the infectious disease.
[0052]
When performing in situ hybridization, first, a detection DNA probe-containing solution (probe solution) prepared using a probe diluent is applied to the smear site, and is applied at about 25 ° C to about 50 ° C, preferably at about 37 ° C. Let stand for about 1 to about 3 hours, preferably about 2 hours, in a humid box at ~ 42 ° C. Thereafter, a hybridization washing solution [a stock solution of hybridization (containing 13.15 g of sodium chloride and 6.615 g of trisodium citrate dihydrate, adjusted to a total volume of 75 ml with sterile purified water), hereinafter simply referred to as "HI The mixture is referred to as “hybridization stock solution”), prepared by mixing hybridization stock solution: sterilized purified water: formamide at a ratio of 5:45:50] in three staining bottles. Soak at 45 ° C., preferably about 42 ° C., for about 10 minutes each. Then, while being immersed in PBS, shake on a shaker for about 5 to about 30 minutes. Specifically, the probe dilution contains 600 μl of salmon sperm DNA, 50 μl of 100 × Denhardt's solution, 500 μl of the above-mentioned hybridization stock, 2250 μl of formamide, and 1000 μl of 50% dextran sulfate. The probe solution preferably contains 15 ng of each detection DNA probe, and the total volume is preferably 50 μl with a probe diluent.
[0053]
The probe concentration of Haemophilus influenzae is about 0.1 to about 2.4 ng / μl, preferably about 0.6 to about 1.8 ng / μl, more preferably about 0.6 to about 1.2 ng / μl. I do.
[0054]
The hybridization test time is at least about 30 minutes or more, preferably about 60 minutes or more, and more preferably about 90 minutes or more. Most preferably, the hybridization time should be set between 120 minutes and 900 minutes.
[0055]
In performing in situ hybridization, it is preferable to use a surfactant such as sodium dodecyl sulfate (SDS) from the viewpoint of increasing the detection sensitivity. A preferred concentration of SDS is about 1% or less, preferably about 0.1% to about 0.75%, more preferably about 0.25% to about 0.5%. SDS has only to be added to the solution used for hybridization, and may be used by being mixed in advance with a probe diluent or a probe solution.
[0056]
The DNA probe may be one or more nucleic acid fragments having a length of about 350 to about 600 bases, preferably about 350 to about 550 bases, and most preferably about 350 to about 500 bases. Is preferred. This is because it facilitates the introduction of the probe into the phagocytes and allows for reliable contact of the incorporated foreign microorganism with the gene. Incidentally, the base length of the probe does not necessarily mean that the base length must fall within the above-described base length range, and it is sufficient that the base length in the above range is included in the probe base length distribution. These probes may be used alone or in combination of several (one or more). The one or more kinds of probes may be a plurality of kinds of probes capable of hybridizing to one strain, and one probe for one strain, but a plurality of species exist. Therefore, the type of the probe may be plural, and there is no particular limitation as long as the type of the probe is one or more.
[0057]
The probe preferably contains a DNA fragment having a sequence with poor cross-reactivity with the phagocyte itself (does not hybridize), and hybridizes with a gene derived from another strain different from the source species of the probe. It must not be.
[0058]
For example, if a subtraction method is used, a specific probe can be created in a short time. These probes should be prepared and labeled according to a standard nick translation method using a non-radioactive isotope labeling substance such as fluorescein isothiocyanate (FITC), biotin, digoxigenin (digoxigenin (DIG) -11-dUTP). Can be. The chain length of the probe can be controlled so that labeling can be performed most efficiently by changing the quantitative ratio of DNase I and DNA polymerase I added in the nick translation reaction.
[0059]
As an example, in order to efficiently label 2 μg of the probe HI-21-8 of the present invention and to make the probe chain length (350 to 600) capable of in situ hybridization with a foreign microorganism DNA efficiently, the total amount is 100 μl. For 2 μl of 10 U / μl of DNA polymerase I in a reaction solution of about 10 to about 350 mU, preferably about 25 to about 200 mU, more preferably about 50 to about 150 mU in a total volume of 100 μl reaction solution The prepared DNaseI is prepared so as to be present in an amount of 6 μl. In this case, the volume of each enzyme and the total amount of the reaction solution may be appropriately changed as long as the ratio of the above-mentioned optimum reaction conditions is constant. In other words, for 20 U of DNA polymerase I in a total volume of 100 μl, adjust the concentration of DNase I to a concentration of about 10 to about 350 mU, preferably about 25 to about 200 mU, more preferably about 50 to about 150 mU. I do. In other words, about 0.5 / 1000 to about 17.5 / 1000, preferably about 1.25 / 1000 to about 10/1000, more preferably about 2/100 DNA polymerase I per unit. It is desirable to perform the nick translation reaction using 0.5 / 1000 to about 7.5 / 1000 units of DNaseI. Further, when focusing on 1 μg of DNA, DNase I should be about 5 to about 175 mU, preferably about 12.5 to about 100 mU, and more preferably about 25 to about 75 mU for 10 U of DNA polymerase I. Good. For other probes, the optimal reaction conditions for the amount of DNA, DNA polymerase I and DNase I can be determined with reference to the above-mentioned optimal reaction conditions. In addition, it can be adjusted to a probe chain length (about 350 to about 600 bases) that allows efficient labeling and allows efficient in situ hybridization with foreign microorganism DNA.
[0060]
By the way, “stringent conditions” used when performing in situ hybridization include, for example, about 30% to about 60%, preferably about 30% to about 50 ° C. in the presence of about 50% formamide. Preferably, the incubation is carried out at about 38 to about 42 ° C., followed by washing.
[0061]
After performing in situ hybridization, a blocking step can also be added. Specifically, 1 ml of a blocking reagent (containing 2 ml of normal rabbit serum and 0.5 ml of PBS stock solution and adjusted to a total volume of 10 ml with sterile purified water) per slide glass in a wet box is dropped on the smear site. And let stand for about 15 to about 60 minutes. Thereafter, the blocking reagent is removed.
[0062]
Hybridization signal detection
In order to detect a signal resulting from hybridization with a gene (genomic DNA or RNA) derived from a bacterium, a color reaction using a standard antigen-antibody reaction or the like is performed.
[0063]
That is, after sufficiently washing the sample after hybridization, a blocking treatment is performed, and then a conjugate such as an anti-FITC antibody or an anti-digoxigenin antibody, for example, a treatment using an alkaline phosphatase conjugate is performed. A signal is developed using a color development system (1) to check the hybridization status. For example, when a probe labeled with digoxigenin-11-dUTP is used as the probe, an anti-digoxigenin-alkaline phosphatase conjugate is used, and substrates for commonly used alkaline phosphatase (nitro blue tetrazolium and 5-bromo-4-chloro-) are used. 3-indolyl phosphate).
[0064]
The smear prepared by washing after the color reaction is counterstained with naphthol black, Fast Green (20 mg / 50 ml, manufactured by Wako Chemicals), etc., and intracellular signals are observed by microscopic examination with an optical microscope. .
[0065]
Specifically, to obtain a signal by hybridization, for example, when a digoxigenin-labeled DNA probe is used as a detection DNA probe, a labeled antibody (1.05 units of an alkaline phosphatase-labeled anti-digoxigenin antibody solution, buffer A (triethanol Amine containing 746 mg of amine, 17.5 mg of sodium chloride, 20.3 mg of magnesium chloride hexahydrate, 1.36 mg of zinc chloride, 1000 mg of bovine serum albumin and an appropriate amount of hydrochloric acid, and adjusted to a total volume of 100 ml with an appropriate amount of sterilized purified water. ) A labeled antibody diluent (8.48 mg of tris- (hydroxymethyl) -aminomethane, 6.14 mg of sodium chloride and an appropriate amount of hydrochloric acid, and an appropriate amount of sterilized purified water) prepared by adjusting the total amount to 14 μl with 12.6 μl. To 0.7ml The prepared labeled antibody solution is diluted about 10-fold to about 200-fold, preferably about 50-fold, and 10 μl of the diluted solution is dropped onto the smear site and left to stand for about 15 to about 60 minutes. Then, the labeled antibody washing solution (containing 1 ml of polysorbate 20 and 50 ml of undiluted PBS solution and adjusted to a total volume of 100 ml with sterile purified water) was diluted about 2 to 50 times, preferably about 10 times. Immerse in the solution and shake it as it is on a shaker for about 5 to about 30 minutes. After this operation was repeated twice, the color pretreatment liquid-1 (containing 6.06 g of tris- (hydroxymethyl) -aminomethane, 2.92 g of sodium chloride and an appropriate amount of hydrochloric acid, and was completely diluted with an appropriate amount of sterilized purified water. Was adjusted to 50 ml) and an equal amount of pre-coloring solution-2 (containing 5.08 g of magnesium chloride hexahydrate and adjusted to a total volume of 50 ml with sterilized purified water), and then mixed with sterilized purified water. And then immersed in a pre-coloring solution diluted about 5 times with a shaking machine for about 5 to about 30 minutes. Thereafter, 1 ml of a coloring reagent (nitro blue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolyl phosphate (BCIP)) per slide glass was dispensed with a disposable syringe equipped with a 0.2 μm syringe top filter. While filtering, the solution is dropped on the smeared portion of the slide glass, and left in a wet box at about 10 ° C. to about 45 ° C., preferably about 37 ° C. for about 15 to about 60 minutes. Thereafter, a coloring reagent washing solution (containing 606 mg of tris- (hydroxymethyl) -aminomethane, 186 mg of disodium ethylenediaminetetraacetate dihydrate and an appropriate amount of hydrochloric acid, and adjusted to a total volume of 50 ml with an appropriate amount of sterilized purified water) Is soaked in a solution diluted about 2 to about 50 times, preferably about 10 times, for about 2 to about 10 minutes, air-dried, and then a counterstain solution (including 50 mg of Fast Green FCF (edible green No. 3), and A solution prepared by diluting the total volume to 50 ml with an appropriate amount of sterile purified water) from about 2 to about 50 times, preferably about 10 times, and about 0.1 to about 5%, preferably about 1 to 10 times. Soak in a solution of acetic acid at 10%. Thereafter, the above-described washing solution of the coloring reagent is soaked again in a solution diluted to about 2 to about 50 times, preferably about 10 times, to wash away the excess counterstaining solution and completely air-dried. Further, the coloring reagent may be a reagent prepared individually.
[0066]
An alkaline phosphatase-labeled anti-digoxigenin antibody solution was prepared by blotting 1 ng of digoxigenin-labeled DNA on a blotting membrane, blocking, treating with a 10,000-fold diluted alkaline phosphatase-labeled anti-digoxigenin antibody solution, and forming a chromogenic substrate (NBT / When BCIP) is reacted, the DNA blotting site develops color, and it is desirable to use a DNA that does not develop color even if the same operation is performed on DNA not labeled with digoxigenin. The anti-digoxigenin antibody is preferably one derived from sheep. In particular, it is preferable to purify from immunized sheep serum through ion exchange chromatography and antibody column chromatography.
[0067]
As a coloring reagent (NBT / BCIP solution, pH 9.0 to 10.0), nitrotetrazolium blue (NBT) 3.3 mg, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) 1.65 mg, It contains 99 μg of N, N-dimethylformamide, 121 mg of tris (hydroxymethyl) aminomethane, an appropriate amount of hydrochloric acid, 58.4 mg of sodium chloride, and 101.6 mg of magnesium chloride hexahydrate, and is completely dissolved in an appropriate amount of sterilized purified water. Those prepared to 10 ml are preferred.
[0068]
As the color-forming reagent, a reagent in which a protein labeled with alkaline phosphatase is blotted on a blotting membrane, and the membrane is treated with a color-forming reagent at room temperature under light-shielding, and a dark purple signal appears at the blot site is preferable.
[0069]
When such counterstaining is performed, an edible dye, for example, yellow No. 4 (tartrazine) can be used to further clarify the signal-cell contrast. The reason for this is that if the developed colors are similar to each other, such as purple coloration by the substrate and blue color by naphthol black, clear counterstaining is difficult to perform. When an edible colorant that had not been tested was used, the difference in hue at the time of counterstaining was clarified and proved to be practical.
[0070]
By the way, as a method for labeling digoxigenin, a nick translation method can be used. As other methods for labeling digoxigenin, a PCR method, a random primer labeling method, an in vitro transcription labeling method, a terminal transferase labeling method, and the like can also be used.
[0071]
Judgment of the presence or absence of cross-reactivity is based on microscopic examination (× 1,000) using a light microscope, in which a single well shows any blue-violet color in cells stained with a counterstain. Is determined to be positive.
[0072]
Also, the method of preparing the detection probe will be apparent by referring to Japanese Patent No. 2965644.
[0073]
Furthermore, to cultivate the probe HI-21-8 of the present invention by fishing from a working cell bank of Haemophilus influenzae HI-21-8, for example, a platinum loop or a disposable plastic loop or the like is used. The bacteria are picked and streaked on a solid L-broth medium containing 50 μg / ml ampicillin placed in a sterile petri dish. After overnight culture, a single colony is collected, inoculated into 5 ml of an L-broth medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. overnight with shaking (preculture). Next, 2.5 ml of the preculture solution is inoculated into a culture flask containing 400 ml of the liquid medium described above, and cultured with shaking at about 37 ° C. overnight (main culture).
[0074]
Then, in order to extract plasmid DNA of Haemophilus influenzae HI-21-8, the culture solution obtained by main culture is centrifuged at about 4,000 × g for 10 minutes at about 4 ° C. to collect the bacteria. The culture supernatant was removed, and 20 ml of STE (10 mmol / l tris-hydrochloric acid (pH 8.0), 1 mmol / l ethylenediamine-tetraacetic acid disodium salt (EDTA), 0.1 mmol / l sodium chloride) was added, and the cells were added. Resuspend and centrifuge at 4,000 xg for 10 minutes at about 4 ° C to collect the cells. 5 ml of solution-1 (50 mmol / l glucose, 25 mmol / l tris-hydrochloric acid (pH 8.0), 10 mmol / l EDTA) containing 10 mg / ml lysozyme is added, and the cells are suspended and left at room temperature for 5 minutes. 10 ml of Solution-2 (0.2 mmol / l sodium hydroxide, 1% sodium dodecyl sulfate (SDS)) is added, mixed by inversion, and left on ice for 10 minutes. Add 7.5 ml of ice-cooled solution-3 (3 mol / l potassium acetate (pH 4.8)), mix by inversion and leave on ice for 10 minutes. After centrifugation at about 45,000 × g for 30 minutes at about 4 ° C. using a high-speed cooling centrifuge, the supernatant is collected and allowed to reach room temperature. Then, add 0.6 volume (about 24 ml) of isopropanol, mix by inversion, and leave at room temperature for 15 minutes or more. After centrifugation at about 28,000 × g for 30 minutes at about 25 ° C. using a high-speed cooling centrifuge, the supernatant is discarded, and the pellet is washed with 70% ethanol and air-dried. After air-drying, 8 ml of TE (10 mmol / l Tris-HCl (pH 8.0), 1 mmol / l EDTA) is added and dissolved (extraction of plasmid DNA).
[0075]
Next, the plasmid DNA containing Haemophilus influenzae HI-21-8 is purified. To the obtained plasmid DNA, 800 μl of 10 mg / ml ethidium bromide and 8.6 g of cesium chloride are added, and mixed by inversion to dissolve. Put the lysate in an ultracentrifuge tube and cap or seal. After ultracentrifugation at 500,000 × g for 5 hours at about 20 ° C. with a vertical rotor, the band of plasmid DNA is collected using a syringe or a needle under irradiation with ultraviolet light. An equal amount of TE-saturated 1-butanol is added to the separated plasmid DNA solution, mixed by inversion, and centrifuged at about 15,000 × g for 5 minutes using a micro high-speed centrifuge to remove the supernatant. This operation is repeated to remove ethidium bramide in the plasmid DNA solution. Next, TE is added to make a volume of 1.5 ml, and desalting is performed with a desalting column (NAP-10). To the desalted plasmid DNA solution, 30 μl of a 3 mol / l sodium acetate solution is added and mixed, and then 3 times the amount of 99.5% ethanol is added, and the mixture is inverted, and left at −20 ° C. for 30 minutes or more. Then, the mixture is centrifuged at 15,000 × g for 20 minutes at 4 ° C. using a microcooled high-speed centrifuge to remove the supernatant, and then suspended by adding cold 70% ethanol. Then, the mixture is again centrifuged at 15,000 × g for 20 minutes at 4 ° C. using a microcooled high-speed centrifuge to remove the supernatant, and the plasmid DNA precipitate is dried under reduced pressure. The plasmid DNA is completely dissolved by adding 100 μl of TE, and the concentration is measured by absorbance at 260 nm (purification of plasmid DNA containing Haemophilus influenzae HI-21-8). Then, the plasmid DNA containing Haemophilus influenzae HI-21-8 is treated with a restriction enzyme, and the size of HI-21-8 is checked by agarose electrophoresis.
[0076]
Next, the probe HI-21-8 is purified through restriction enzyme treatment of the plasmid DNA containing Haemophilus influenzae influenzae probe HI-21-8 and agarose electrophoresis. For this purpose, 1 mg of the plasmid DNA containing the probe HI-21-8, whose molecular weight has been confirmed, is allowed to proceed at 37 ° C. for 1.5 hours or more at 37 ° C. by using the restriction enzyme HindIII alone or in combination with another restriction enzyme. Digestion by
[0077]
After digesting the plasmid DNA, a part of the reaction solution is electrophoresed on 0.8% agarose to confirm that the digestion has been completed. After confirming the digestion, the sample is subjected to electrophoresis on a preparative 0.8% agarose gel to collect the probe HI-21-8 band. The collected probe HI-21-8 is extracted and purified from an agarose gel, and its concentration is measured with an absorptiometer.
[0078]
A part of the purified probe HI-21-8 is electrophoresed on a 0.8% agarose gel to confirm that it is a single band.
[0079]
Next, in order to label the probe HI-21-8, 2 μg of the purified HI-21-8 is labeled with digoxigenin in a reaction solution having the composition shown in Table 1 below.
[0080]
[Table 1]

[0081]
In Table 1, “X” refers to a volume that can be added to a preferred probe concentration in accordance with the concentration of the probe stock solution, and the final volume is adjusted by determining the purified water amount Y in accordance with this volume.
[0082]
After labeling, the reaction is stopped by adding 100 μl of TE to the reaction solution. The reaction stop solution is injected into a spin column and centrifuged at about 380 × g for about 10 minutes at about 4 ° C. to remove free nucleotides. Next, the concentration of the eluate is measured with an absorptiometer, and adjusted to about 10 ng / μl with TE.
[0083]
To confirm the labeling, 0.5 μl of the labeled probe HI-21-8 is dropped on the membrane and air-dried. This membrane is immersed in a blocking reagent and blocked at room temperature for 30 minutes. The membrane is immersed in an alkaline phosphatase-labeled anti-digoxigenin antibody solution diluted 5,000-fold with 0.1 mol / l Tris-HCl (pH 7.5) and 0.15 mol / l sodium chloride at room temperature for 30 minutes. The membrane is immersed in 0.1 mol / l tris-hydrochloric acid (pH 7.5) and 0.15 mol / l sodium chloride, and washed twice by shaking at room temperature for about 10 minutes. The membrane is immersed in 0.5 mol / l tris-hydrochloric acid (pH 9.5), 0.15 mol / l sodium chloride, and 50 mmol / l magnesium chloride at room temperature for about 10 minutes.
[0084]
The membrane is immersed in the coloring reagent at room temperature and protected from light for about 10 minutes. The membrane is immersed in TE to stop coloring. Confirmation of labeling with blue-violet color under the spot.
[0085]
A 1 ml disposable syringe is filled with a small amount of sterilized glass wool to make a spin column. 1 mmol / l Tris-HCl (pH 7.5), 1 mmol / l EDTA, Sephadex G-50 swollen with 0.1% SDS are filled in a syringe. The syringe is placed in a 15 ml disposable conical tube, and centrifuged at about 320 × g for about 10 minutes at about 4 ° C. to remove excess buffer. After removing the syringe from the disposable conical tube and discarding the discharged buffer solution, a 1.5 ml Eppendorf type tube is placed at the bottom of the disposable conical tube, and the syringe is placed thereon.
[0086]
Dot blot hybridization
In order to confirm the specificity of the probe, dot blot hybridization is performed according to the following procedure.
[0087]
First, in order to denature each spotted genomic DNA, various bacterial genomes prepared on filter paper (Whatman 3MM) saturated with 0.5 mol / l sodium hydroxide and 1.5 mol / l sodium chloride solution according to a standard method. 100 ng was spotted on a nylon membrane (Paul Biodyne Type B, manufactured by Pall Japan), and the air-dried membrane was allowed to stand for 10 minutes. Next, the denatured DNA is neutralized by allowing to stand on the above-mentioned filter paper saturated with 0.5 mol / l Tris-hydrochloric acid (pH 7.5) and 1.5 mol / l sodium chloride solution for 10 minutes. Further, the filter is allowed to stand on the filter paper saturated with a 2 × SSC (Standard Saline Citrate) solution for 5 minutes and washed.
[0088]
Then, the membrane is air-dried, the membrane is immersed in a 2 × SSC solution, and immersed for 5 minutes. According to the usual method, the membrane is immersed in the prehybridization solution in a plastic bag, and allowed to undergo affinity at 42 ° C. for 60 minutes. The membrane is immersed in 15 ml of a hybridization solution containing 400 ng of the probe in a plastic bag, and reacted at 42 ° C. overnight. Next, the membrane is immersed in a 2 × SSC, 0.1% SDS (sodium lauryl sulfate) solution and washed for 5 minutes (this step is repeated twice). Thereafter, the membrane is immersed in a 0.1 × SSC, 0.1% SDS solution, and washed at 60 ° C. for 10 minutes (this step is repeated three times). Immerse the membrane in 2 × SSC solution and wash for 5 minutes. The membrane is immersed in a solution containing 3% bovine serum albumin, 1% blocking buffer (manufactured by Boehringer), 0.1 mol / l Tris-hydrochloric acid (pH 7.5), and 0.15 mol / l sodium chloride. To shake.
[0089]
Next, the membrane was added to a solution obtained by diluting an alkaline phosphatase-labeled anti-digoxigenin antibody (manufactured by Boehringer) 5,000 times with 0.1 mol / l Tris-HCl (pH 7.5) and 0.15 mol / l sodium chloride solution. Soak and shake gently for 30 minutes. Next, the membrane is immersed in a 0.1 mol / l tris-hydrochloric acid (pH 7.5), 0.15 mol / l sodium chloride solution, and shaken for 15 minutes (this step is repeated twice). The membrane is immersed in a solution containing 0.1 mol / l Tris-hydrochloric acid (pH 9.5), 0.1 mol / l sodium chloride, and 5 mmol / l magnesium chloride, and shaken for 5 minutes. The membrane is immersed in an NBT-BCIP solution (manufactured by GIBCO BRL), and a color reaction is performed under light shielding. The membrane is immersed in TE (10 mmol / l Tris-hydrochloric acid (pH 8.0), 1 mmol / l EDTA) to stop the coloring reaction, and air-dried. The compositions of the pre-hybridization solution and the hybridization solution are shown in Table 2 below.
[0090]
[Table 2]

[0091]
As the surfactant used in the above-mentioned in situ hybridization step, a known surfactant can be used.
[0092]
Surfactants are broadly classified into anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants.
[0093]
The anionic surfactant is also referred to as an anionic surfactant, and is an ionizing substance in water that becomes an organic anion. When the lipophilic group in the molecule of the surfactant is expressed as R, RCOONa, RSO₃Na, RSO₄It is represented by the formula of Na. In those containing a weakly acidic group such as RCOONa, the aqueous solution thereof is easily hydrolyzed and exhibits weak alkalinity.₃Na, RSO₄In the case of a compound having a strong acidic group such as Na, the aqueous solution is less susceptible to hydrolysis and exhibits neutrality. Since it is anionic, it may lose its surface activity in the presence of a large amount of a cationic substance, and also becomes inactive when it is made strongly acidic.
[0094]
The nonionic surfactant refers to a surfactant having a nonionic hydrophilic group. As a hydrophilic group, an ethylene oxide group (-CH₂CH₂O-) is frequently used, and as the number of functional groups increases, hydrophilicity increases. Conversely, as the number of carbon atoms in the lipophilic group increases, the lipophilicity increases.
[0095]
Therefore, it is characterized in that a surfactant having variously changed hydrophilicity and lipophilicity can be obtained. Nonionic surfactants do not ionize in water and are not easily affected by inorganic salts, and therefore have little effect on living bodies. In addition, since it has a strong cleaning action and relatively little foaming, it is used not only for detergents but also for various applications such as pharmaceuticals, cosmetics, and foods. When the temperature increases, the water-soluble nonionic surfactant becomes difficult to dissolve in water at some point, and the aqueous solution becomes cloudy. This occurs because the hydrogen bond between the hydrophilic group and water is broken.
[0096]
Cationic surfactants are also referred to as cationic surfactants, which ionize in water to organic cations. Cationic surfactants generally do not have a large detergency, but have a strong bactericidal action because they strongly bind to anionic substances such as bacteria. It also has antistatic ability with fibers and plastics. A typical cationic surfactant, dodecyltrimethyl chloride [C₁₂H₂₅(CH₃)₃N] Cl is water-soluble, while didodecyldimethylammonium chloride [(C₁₂H₂₅)₂(CH₃)₂N] Cl is hardly soluble in water and forms bimolecular membrane vesicles in water, which are soluble in benzene.
[0097]
The amphoteric surfactant is a surfactant having both an anionic group and a cationic group in a molecule. The ionization state in an aqueous solution is similar to an amino acid, and an amphoteric surfactant contains many amino acid derivatives. Therefore, like an amino acid, it has an isoelectric point and acts as an anionic surfactant when it is on the alkaline side of the isoelectric point, and acts as a cationic surfactant when it is on the acidic side. At the isoelectric point, water solubility is lowest and surface tension is lowest. Amphoteric surfactants are used for applications such as bactericides and antistatic agents.
[0098]
Anionic surfactants are classified into carboxylic acid type, sulfonic acid type, sulfate ester type and phosphate ester type, and nonionic surfactants are classified into ester type, ether type, ester ether type and alkanolamide type. being classified. Cationic surfactants are classified into alkylamine salt type and quaternary ammonium salt type, and amphoteric surfactants are classified into carboxybetaine type, 2-alkylimidazoline derivative type and glycine type.
[0099]
In addition, the carboxylic acid form of the anionic surfactant is subdivided into fatty acid monocarboxylates, N-acyl sarcosine salts and N-acyl glutamates. Typical examples thereof include sodium laurate and medicated soap as fatty acid monocarboxylates, and N-lauroyl sarcosine sodium, N-acyl glutamate, and disodium N-lauroyl glutamate as N-acyl sarcosine salts. is there. The sulfonic acid type includes dialkyl sulfosuccinate, alkane sulfonate, alpha olefin sulfonate, linear alkylbenzene sulfonate, alkyl (branched chain) benzene sulfonate, alkyl naphthalene sulfonate, naphthalene sulfonate -Subdivided into formaldehyde condensate and N-methyl-N-acyltaurine salt. As typical examples, dialkyl sulfosuccinates include sodium dioctyl sulfosuccinate, alkane sulfonates include sodium dodecane sulfonate, linear alkylbenzene sulfonates include linear sodium dodecylbenzene sulfonate, and alkyl (branched chain). Examples of the benzenesulfonate include sodium dodecylbenzenesulfonate, examples of the alkylnaphthalenesulfonate include sodium butylnaphthalenesulfonate, and examples of the N-methyl-N-acyltaurinate include sodium N-methyl-N-stearoyltaurine.
[0100]
The sulfate ester type is subdivided into alkyl sulfates, polyoxyethylene alkyl ether sulfates, and fats and oils sulfates. Typical examples thereof include sodium dodecyl sulfate, sodium lauryl sulfate and sodium cetyl sulfate as alkyl sulfates, and polyoxyethylene lauryl ether triethanolamine as polyoxyethylene alkyl ether sulfate. The phosphate ester type is subdivided into alkyl phosphates, polyoxyethylene alkyl ether phosphates, and polyoxyethylene alkyl phenyl ether phosphates. A typical example thereof is disodium monolauryl phosphate as an alkyl phosphate.
[0101]
Polyoxyethylene alkyl ether phosphates include sodium polyoxyethylene lauryl ether and polyoxyethylene oleyl ether phosphate (8MOL).
[0102]
The ester form of the nonionic surfactant is subdivided into fatty acid glycerin, fatty acid sorbitan and fatty acid sucrose esters. As typical examples, glyceryl monostearate as fatty acid glycerin, sorbitan monostearate, sorbitan trioleate, sorbitan sesquioleate, sorbitan monolaurate, polysorbate 20 (polyoxyethylene sorbitan fatty acid ester), polysorbate as fatty acid glycerin 60 and polysorbate 80, fatty acid sucrose esters include sucrose stearate. The ether type is subdivided into polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether and polyoxyethylene polyoxypropylene glycol. Representative examples thereof include polyoxyethylene lauryl ether, polyoxyethylene stearyl ether and polyoxyethylene cetyl ether as polyoxyethylene alkyl ethers, and polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether and polyoxyethylene alkylphenyl ether. There is ethylene octyl phenyl ether. Further, the ester ether type is subdivided into fatty acid polyethylene glycol and fatty acid polyoxyethylene sorbitan. Representative examples of the fatty acid polyethylene glycol include polyethylene glycol oleate, and the fatty acid polyoxyethylene sorbitan includes polyoxyethylene sorbitan palmitate and polyoxyethylene sorbitan monolaurate. The alkanolamide type is only fatty acid alkanolamide, and lauric acid diethanolamide is a typical example.
[0103]
The alkylamine salt type of the cationic surfactant includes a monoalkylamine salt, a dialkylamine salt and a trialkylamine salt, and monostearylamine hydrochloride is a typical example. Also, the quaternary ammonium salt type is subdivided into alkyltrimethylammonium chloride (or bromide, iodide), dialkyldimethylammonium chloride (or bromide, iodide), and alkylbenzalkonium chloride. As typical examples thereof, stearyltrimethylammonium chloride is used as alkyltrimethylammonium chloride (or bromide or iodide), distearyldimethylammonium chloride is used as dialkyldimethylammonium chloride (or bromide or iodide), and As alkylbenzalkonium, there is laurylbenzalkonium chloride.
[0104]
The only carboxybetaine type of amphoteric surfactant is alkylbetaine, and laurylbetaine is a typical example. The derivative of 2-alkylimidazoline is only 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, and 2-undecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine. Is a typical example. The glycine type includes alkyl (or dialkyl) diethylenetriaminoacetic acid, and a typical example thereof is dioctyldiethylenetriaminoacetic acid.
[0105]
Further, in addition to those listed above, Triton X-100, lauryl sarcosine, saponin, BRIJ35, alkyl allyl polyether alcohol, higher alcohol sulfate, N-cocoyl-L-arginine ethyl ester DL-pyrrolidone carboxylate, N -Cocoyl-sodium N-methylaminoethylsulfonate, cholesterol, self-emulsifying glyceryl monostearate, squalane, stearyl alcohol, polyoxyl stearate 40, cetanol, setocrogol 1000, diethyl sebacate, nonylphenoxypolyoxyethylene ethane sulfate Ester ammonium, polyoxyethylene oleylamine, polyoxyethylene sorbite beeswax, polyoxyl 35 castor oil, macrogol 400, N-coconut oil fatty acid Sil L-arginine ethyl / DL-pyrrolidone carboxylate, lauryl dimethylamine oxide solution, lauromacrogol, methylcellulose, CMC (carboxymethylcellulose), polyoxyethylene hydrogenated castor oil 20 and polyoxyethylene hydrogenated castor oil 60, CHAPS, deoxy Cholic acid, digitonin, n-dodecyl maltoside, nonidet P40, n-octyl glucoside, octyl thioglucoside, sucrose laurate, dodecyl poly (ethylene glycol ether) n, n-dodecyl-N, N-dimethyl-3-ammonio- 1-propane sulfonate and the like can also be used.
[0106]
It is important that the various surfactants described above are used in the in situ hybridization step, and the method of use is not particularly limited. For example, it may be mixed in a probe solution or a probe diluent, or a solution containing a surfactant prepared separately from the probe solution is added before, simultaneously with, or after applying the probe solution to the smear site. Alternatively, those skilled in the art can make appropriate changes.
[0107]
In the present invention, if necessary, a positive control probe can be prepared. For example, first, in order to extract and purify genomic DNA of U937 cells (ATCC CRL-1593.2), a cell culture flask (175 cm) containing RPMI1640 medium (25 ml) was placed in a 5% carbon dioxide gas incubator at 37 ° C.³Culture U937 cells in).
[0108]
The U937 culture solution is placed in a 50 ml centrifuge tube, and centrifuged at 4 ° C. at 220 × g for 10 minutes to collect U937 cells. The cells are suspended and washed with 10 ml of PBS, and centrifuged again at 4 × C at 180 × g for 10 minutes to collect the cells. Thereafter, the supernatant is discarded, and the cells are suspended in 1 ml of a TE solution containing 1% SDS containing 200 μg / ml proteinase K and left at 37 ° C. for 30 minutes. Phenol extraction is repeated 3 to 4 times to remove proteins. The genome precipitated by ethanol precipitation is recovered, dissolved in 500 μl of sterilized purified water containing 2.5 μg ribonuclease, and left at 42 ° C. for 30 minutes. Phenol extraction is repeated 2-3 times to remove proteins. The genome precipitated by ethanol precipitation is collected and dissolved in 500 μl of TE. Thereafter, a positive control probe can be prepared by measuring the concentration with an absorptiometer and applying it to a digoxigenin label. Further, it is preferable to use a positive control probe which can confirm hybridization when dot-hybridizing the positive control probe on a membrane on which 100 ng of U937 genome is spotted.
[0109]
Similarly, if necessary, a negative control probe can be prepared by a known method.
[0110]
Other embodiments
A kit for detecting and / or identifying Haemophilus influenzae using the detection method of the present invention is also provided by the present invention. According to the kit of the present invention, the enzyme (DNA exposure treatment agent) used in the step of obtaining DNA is at least one enzyme selected from the group consisting of at least lysostaphin, lysozyme, N-acetylmuramidase, and zymolase. , A probe solution to which a surfactant has been added, and one or more DNA probes for detection. The kit of the present invention includes a blood separation reagent, an enzyme pretreatment reagent, an enzyme reagent, an acetylation reagent, a probe solution, a blocking reagent, a labeled antibody, a labeled antibody diluent, and a color pretreatment as exemplified in the following Examples. Solution-1, Coloring pretreatment solution-2, Coloring reagent, Counterstaining solution, PBS stock solution, Hybridization stock solution, Labeled antibody washing solution, Coloring reagent washing solution, APS coated slide glass, Probe diluent, Buffer A, etc. It is possible. Of these, it is preferable to include at least an enzyme reagent and a probe solution. Various reagents such as chloroform, ethanol, acetic anhydride, DMSO, PMSF, formamide, acetic acid, hydrochloric acid, and sodium hydroxide can also be used. In addition, low-speed centrifuge, thermostat, hemocytometer, shaker, wet box, thermostat, optical microscope, variable pipette, blood collection tube, tip, pipette, staining bottle, measuring cylinder, syringe, 0.2 μm syringe A device such as a top filter may be provided.
[0111]
According to the present invention, there is provided a method for monitoring a gene of a foreign microorganism phagocytosed by a phagocyte contained in a clinical specimen containing a phagocyte derived from a living body.
[0112]
Further, according to the present invention, there is also provided a method for identifying a gene causing a sepsis or a bacteremia based on a result of identifying a gene of a microorganism which is a candidate of the causative microorganism. According to this method, when applied to the diagnosis of various suspected sepsis patients' blood, it was not affected by the administered antimicrobial agent, and was approximately four times as sensitive as the blood culture method. Can be detected, and it is clear that the coincidence rate of the detected strains is good. In comparison with blood culture, which requires at least 3 days or more, usually about 14 days, for the test, the method of the present invention makes it possible to perform an extremely short and simple operation of about 8 hours to complete the entire operation. Accurate results can be obtained. Therefore, according to this method, a useful marker is provided in the diagnosis of infectious diseases, such as sepsis or bacteremia, which need to be dealt with promptly and appropriately, and for monitoring the prognosis.
[0113]
In addition, if primers are designed with reference to the nucleotide sequence information of the probe of the present invention, the infectious disease-causing bacteria can be identified by amplification of DNA by PCR without hybridization.
[0114]
Further, it is clear that the probe of the present invention or a fragment thereof can be used by being incorporated into a DNA chip. Then, the presence of Haemophilus influenzae can be determined using the DNA chip.
[0115]
In addition, the nucleotide sequence disclosed in the present invention is obtained by randomly cloning genomic DNA of Haemophilus influenzae, and therefore, the usefulness of the nucleotide sequence of the present invention extends to its complementary strand. It is. Furthermore, it is naturally conceivable that a mutated portion is present in the DNA of the wild-type strain, but generally, it is about 70%, preferably 70% or more, more preferably 80% or more, and still more preferably the probe of the present invention. Since a nucleotide sequence having 90% or more homology is considered to be able to achieve the object of the present invention, the present invention naturally includes these homologous sequences.
[0116]
A. Haemophilis Haemophilus influenzae detection and identification
Specific examples of procedures for the general detection and identification of Haemophilus influenzae from clinical specimens are detailed below.
[0117]
(1) Blood collection and blood sample processing
A blood sample collected from a patient suspected of sepsis is used as a clinical sample. 10 ml of heparinized venous blood was collected from each patient, and the collected blood and a blood separation reagent (225 mg of sodium chloride, 1.5 g of dextran (molecular weight: 200,000 to 300,000), and a total volume of 25 ml were prepared using sterilized purified water. ) Were mixed at a ratio of 4: 1 and left at 37 ° C. for 30 minutes to obtain a leukocyte fraction (upper layer). The leukocyte fraction thus obtained is centrifuged at 4 ° C. and 160 × g for 10 minutes to obtain leukocytes. Next, 1 ml of sterile purified water was added to the obtained leukocyte pellet to suspend the suspension. Immediately, an excess amount of PBS (18.24 g of sodium chloride, 6.012 g of sodium monohydrogen phosphate decahydrate, 6.012 g of diphosphate) was added. After adding 1.123 g of sodium hydrogen dihydrate, made up to a total volume of 120 ml with sterile purified water (diluted PBS solution 20-fold with sterile purified water), the solution was made isotonic, and again at 4 ° C. Then, centrifuge at 160 × g for 10 minutes.
[0118]
(2) Leukocyte fixation
An APS-coated slide glass in which 3-aminopropyltriethoxysilane (APS, SIGMA) is coated on a slide glass (product number MS311BL, manufactured by Air Brown Japan) is used.
[0119]
In preparing an APS-coated slide glass, first, a slide glass (MS311BL) is fixed to a slide holder, and then immersed and washed in a diluted neutral detergent for 30 minutes or more, and the detergent is sufficiently removed with tap water. The slide glass is washed with purified water, sufficiently dried at a high temperature (100 ° C. or higher), and then left to cool at room temperature. Thereafter, the slide glass is immersed in acetone containing 2% APS for 1 minute, immediately washed lightly with acetone and sterile purified water, and then air-dried. Furthermore, the slide glass is immersed again in acetone containing 2% APS for 1 minute, and immediately washed lightly with acetone and sterile purified water. Then, after performing an operation of air-drying, it is dried at 42 ° C. to produce an APS-coated slide glass.
[0120]
A small amount of PBS is added to and suspended in the leukocyte pellet obtained by centrifuging the leukocyte fraction at 160 × g for 10 minutes at 4 ° C., and the number of leukocytes is counted using a hemocytometer.
[0121]
1 × 10 cells⁵5 μl of a leukocyte suspension prepared in PBS to give cells / well was smeared onto each well of an APS-coated slide glass so that the leukocytes spread in a monolayer, and completely air-dried, whereby the leukocytes were coated on the APS-coated slide glass. To be supported.
[0122]
Then, the plate is immersed in a Carnoy's fixative (fixed solution obtained by mixing ethanol: chloroform: acetic acid = 6: 3: 1) for 20 minutes, then immersed in a 75% ethanol solution for 5 minutes, and completely air-dried.
[0123]
(3) Permeability-enhancing treatment of leukocyte cell membrane
After immersion in PBS for 10 minutes, an enzyme pretreatment reagent (saponin 1.25 g, t-octylphenoxypolyethoxyethanol (specific gravity 1.068 to 1.075 (20/4 ° C), pH (5 w / v%)) 5-7.5) 1.25 ml and a PBS stock solution (25 ml) were mixed, and the total volume was adjusted to 50 ml with sterilized purified water). The resultant was immersed in a solution diluted 10-fold with sterilized purified water, and shaken with a shaker for 10 minutes. Let it go.
[0124]
(4) Lysis enzyme treatment of bacterial cell wall
An enzyme reagent solution (in PBS) per slide glass in an enzyme reagent (200 units / ml zymolase, 1,000 units / ml N-acetylmuramidase, 100,000 units / ml lysozyme and / or 100 units / ml lysostaphin) 0.1 mol / l phenylmethylsulfonylfluoride (PMSF) -containing dimethylsulfoxide (DMSO) diluted 100-fold) (1 ml) is added to prepare an enzyme reagent solution. Then, in order to obtain the DNA of the causative bacteria of the infectious disease, 1 ml of this enzyme test solution was dropped on the leukocyte smear site in a humid chamber at 37 ° C. to 42 ° C., and allowed to stand for 30 minutes to obtain the DNA of the causative causative bacteria. To expose. Then, the cells were immersed in PBS containing 0.2 mol / l hydrochloric acid (prepared by adding hydrochloric acid to a PBS stock solution and diluting 20-fold with sterile purified water to adjust the final concentration of hydrochloric acid to 0.2 mol / l). Shake on top for 10 minutes.
[0125]
(5) Acetylation of cell membrane proteins
Acetic anhydride was added to an acetylation reagent (containing 7.46 g of triethanolamine and an appropriate amount of hydrochloric acid, and adjusted to a total volume of 50 ml with an appropriate amount of sterile purified water), and diluted 10-fold with sterile purified water to obtain a final concentration of acetic anhydride. The slide glass is immersed in an acetylation reagent adjusted to 0.8%, and shaken on a shaker for 10 minutes. Then, it is immersed in 75%, 85%, and 98% ethanol sequentially for 3 minutes, and completely air-dried.
[0126]
(6) Fungi DNA Alkali treatment [double-stranded DNA The single strand DNA Denatured]
The slide glass is immersed in PBS containing 70 mmol / l sodium hydroxide (sodium hydroxide added to a stock solution of PBS and diluted 20-fold with sterile purified water to adjust the final concentration of sodium hydroxide to 70 mmol / l) for 3 minutes. . Then, it is immersed in 75%, 85%, and 98% ethanol sequentially for 3 minutes, and completely air-dried.
[0127]
(7) Hybridization
A solution containing 15 ng of digoxigenin-labeled DNA probe prepared in a probe diluent (containing 0.5% SDS, 600 μl of salmon sperm DNA, 50 μl of 100 × Denhardt's solution, 500 μl of hybridization stock solution, 2250 μl of formamide, and 1000 μl of 50% dextran sulfate). (Probe solution, 1.0 ng / μl) is applied to the smear site and allowed to stand in a wet box at 37 ° C. to 42 ° C. for 2 hours. The digoxigenin-labeled DNA probe is prepared by the nick translation method. Thereafter, a hybridization washing solution (hybridization stock solution (13.15 g of sodium chloride, 6.615 g of trisodium citrate dihydrate, prepared with sterilized purified water to a total volume of 75 ml,Above) Was prepared by mixing hybridization stock solution: sterilized purified water: formamide at a ratio of 5:45:50) in three staining bottles, and immersed at 42 ° C. for 10 minutes. Then, it is immersed in PBS and shaken as it is on a shaker for 10 minutes.
[0128]
The nick translation method is used to label the probes HI-21-8, HI-57-34, HI-63-43 and HI-65-39 with digoxigenin.
[0129]
(8) blocking
After performing in situ hybridization, a blocking operation is performed.
[0130]
Specifically, 1 ml of a blocking reagent (containing 2 ml of normal rabbit serum and 0.5 ml of PBS stock solution and adjusted to a total volume of 10 ml with sterile purified water) per slide glass in a wet box is dropped on the smear site. This is left for 30 minutes. Thereafter, the blocking reagent is removed.
[0131]
(9) Reaction with labeled antibody
Labeled antibody (Alkaline phosphatase-labeled anti-digoxigenin antibody solution 1.05 unit, buffer A (triethanolamine 746 mg, sodium chloride 17.5 mg, magnesium chloride hexahydrate 20.3 mg, zinc chloride 1.36 mg, bovine serum albumin 1000 mg) A suitable amount of sterilized purified water containing a suitable amount of hydrochloric acid and adjusted to a total volume of 100 ml) A solution prepared by adjusting the total volume to 14 μl with 12.6 μl) and a labeled antibody diluent (Tris- (hydroxymethyl) -aminomethane 8.48 mg) , Sodium chloride, 6.14 mg and an appropriate amount of hydrochloric acid, and adjusted to a total volume of 0.7 ml with an appropriate amount of sterilized purified water) to prepare a labeled antibody solution 50-fold diluted, and apply 10 μl of the labeled antibody solution to the smear site. The mixture is added dropwise, and this is left for 30 minutes. Then, the labeled antibody washing solution (containing 1 ml of polysorbate 20 and 50 ml of undiluted PBS solution and adjusted to a total volume of 100 ml with sterile purified water) is immersed in a 10-fold diluted solution, and shaken as it is on a shaker for 10 minutes. I let it. After this operation was repeated twice, the color pretreatment solution-1 (containing 6.06 g of tris- (hydroxymethyl) -aminomethane, 2.92 g of sodium chloride and an appropriate amount of hydrochloric acid, and the total amount was 50 ml with an appropriate amount of sterilized purified water. ) And an equal amount of pre-coloring solution-2 (containing 5.08 g of magnesium chloride hexahydrate and adjusted to a total volume of 50 ml with sterile purified water), and mix 5 times with sterile purified water. It is immersed in the diluted color pretreatment solution and shaken as it is on a shaker for 10 minutes.
[0132]
(10) Signal detection
Coloring reagent [nitro blue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution per slide glass, pH 9.0 to 10.0: 3.3 mg NBT, BCIP 1 .65 mg, N, N-dimethylformamide 99 μg, tris (hydroxymethyl) aminomethane 121 mg, an appropriate amount of hydrochloric acid, 58.4 mg of sodium chloride and 101.6 mg of magnesium chloride hexahydrate. Prepared to 10 ml] While filtering using a disposable syringe equipped with a 0.2 μm syringe top filter, 1 ml was dropped onto the smeared portion of the slide glass, and allowed to stand at 37 ° C. for 30 minutes in a wet box at 37 ° C. I do. Thereafter, 10 parts of a coloring reagent washing solution (containing 606 mg of tris- (hydroxymethyl) -aminomethane, 186 mg of disodium ethylenediaminetetraacetate dihydrate and an appropriate amount of hydrochloric acid, and adjusted to a total volume of 50 ml with an appropriate amount of sterilized purified water) was added. After immersing in a 1: 1 diluted solution for 5 minutes and air-drying, a counterstain solution (50 mg of Fast Green FCF (edible green No. 3), prepared to a total volume of 50 ml with sterilized purified water) was diluted 10 times with 1 solution. Soak in a solution of acetic acid at 10% Thereafter, the color reagent washing solution is immersed again in a 10-fold diluted solution to wash away the excess counterstain solution and completely air-dried.
[0133]
(11) Judgment
Judgment was made when microscopic examination (× 1,000) with an optical microscope showed that even if at least one blue-violet coloring signal was observed in cells stained with the counterstain in a single well. Positive ".
[0134]
B. Haemophilis from phagocytic samples Haemophilus influenzae detection
Examples of common procedures for detection of Haemophilus influenzae from engulfed samples are detailed below.
[0135]
(1) Preparation of phagocytic sample
(I) Preparation of U937 cells
A cell culture flask (175 cm) containing RPMI 1640 medium (25 ml) in a 5% carbon dioxide incubator at 37 ° C.³)), Culture U937 cells (human monocyte cell line, ATCC CRL-1593.2). Next, the U937 cell culture solution is placed in a 50 ml centrifuge tube, and centrifuged at 4 ° C. and 220 × g for 10 minutes to collect U937 cells. Thereafter, the collected U937 cells were suspended in 200 μl of PBS, and the number of cells was calculated using a hemocytometer.⁴Pieces / μl to about 2 × 10⁴Make up to pcs / μl.
[0136]
(Ii) Preparation of Bacterial Phagocytosis Sample
Haemophilus influenzae is inoculated into 5 ml of a BHI culture solution and cultured at 37 ° C. for 8 hours or more. The cultured bacterial solution is centrifuged at 2,000 × g for 10 minutes at 4 ° C. to collect the bacteria. After discarding the supernatant, the bacterial pellet is suspended in 5 ml of PBS, and centrifuged again at 2,000 × g at 4 ° C. for 10 minutes to collect the bacteria. The collected bacteria were suspended in 5 ml of PBS, diluted with PBS, and the turbidity (OD = 600 nm) of the bacterial solution was adjusted to 0.016 to 0.024 by an absorptiometer. Prepare 15 ml. The bacterial solution thus obtained is 175 cm³And left at room temperature for 30 minutes.
[0137]
50 ml of heparinized healthy human blood is collected, a blood cell separation reagent is added at a ratio of 4: 1, the mixture is allowed to stand at 37 ° C. for 30 minutes, a leukocyte fraction is collected, and this is made up to 50 ml with PBS. The supernatant in the culture flask is gently discarded, the leukocyte fraction diluted with PBS is added to the flask in 10 ml portions, and the flask is allowed to stand at room temperature for 10 minutes. The supernatant in the culture flask is discarded, and the leukocytes adhered to the bottom of the flask are collected in a 15 ml centrifuge tube with 10 ml of 0.02% EDTA-containing PBS, and centrifuged at 4 ° C. and 140 × g to 180 × g for 10 minutes. And collect white blood cells. When erythrocytes were found to be contaminated in the collected leukocytes, the leukocyte sediment was gently suspended and lysed with 1 ml of sterilized purified water, and then hemolyzed with 14 ml of PBS. Centrifuge at xg-180 xg for 10 minutes to collect leukocytes. The collected leukocytes were suspended in PBS, and the number of cells was counted using a hemocytometer.⁴Pieces / μl to 5 × 10⁴Make up to pcs / μl.
[0138]
The phagocytic sample thus obtained is referred to as an HI phagocytic sample.
[0139]
The advantages of the phagocytic sample are that (1) it is difficult to obtain a clinical sample from which Haemophilus influenzae is detected, so that various tests such as specificity evaluation of the probe of the present invention cannot be performed. (2) that it can be used for specificity tests, sensitivity tests, reproducibility tests, and other performance tests of the probe or kit of the present invention, and (3) phagocytosis. Various test results obtained from the sample can be applied to clinical specimens, and (4) it can be used for setting various conditions such as temperature conditions, reaction time conditions, and enzyme treatment conditions.
[0140]
(Iii) Smear fixation
The U937 cells obtained in (i) and the HI phagocytic sample prepared in (ii) are spread on each well of an APS-coated slide glass in an amount of 5 μl and air-dried.
[0141]
Next, the slide glass was immersed in Carnoy's fixative for 20 minutes, immersed in 75% ethanol for 5 minutes, washed with Carnoy's fixative, air-dried, and stored at 4 ° C. until used for the test (A (2 ))). Next, the pretreatment of the fixed sample is performed according to the procedure described in item A (3).
[0142]
(2) Specifications and test methods for phagocytic samples
(I) Cell count
The number of cells smeared and fixed on the slide glass of the bacteria-phagocytic sample was about 5.0 × 10⁴~ 2.5 × 10⁵Cells / well, and the cell number of U937 cells was about 5.0 × 10⁴~ 1.0 × 10⁵Number / well.
[0143]
(Ii) Devour rate
A bacterial phagocytic sample smeared and fixed on a slide glass is stained with an acridine orange staining solution, and about 200 cells are randomly counted using a fluorescence microscope (× 1,000).
[0144]
Among the measured cells, cells that phagocytose bacteria in the cells (cells in which a morphological change characteristic of phagocytosis is recognized) are defined as positive cells, and the phagocytosis rate is calculated according to the following formula.
[0145]
(Equation 1)

[0146]
(3) Test method
The prepared phagocytic sample is used as a specimen. The specificity of each probe is examined using a slide glass smeared with the used HI digested sample.
[0147]
(4) Result
Together with the probes HI-21-8, HI-57-34, HI-63-43 and HI-65-39, it is found that the probe specifically hybridizes with DNA derived from Haemophilus influenzae taken into phagocytic cells. . Further, according to the mixed probe (including two or more of the probes HI-21-8, HI-57-34, HI-63-43 and HI-65-39), a stronger signal can be obtained as compared with the single probe. It turns out that. For this reason, it is preferable to use a mixture of plural kinds of probes.
[0148]
Since the results obtained with phagocytic samples can be applied to clinical specimens, the probes of the present invention prove to be useful also in clinical specimens.
[0149]
【Example】
BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below in detail and specifically with reference to examples, but it should be understood that the present invention should not be construed as being limited based on the disclosure of these examples.
[0150]
Example 1: Haemophilis Preparation of influenzae detection probe
The clinical strain Haemophilus influenzae was cultured overnight on Chocolate II agar medium (manufactured by Nippon Becton Dickinson), the cultured cells were collected, and N-acetylmuramidase SG was added in the lysis step. A modified method of Saito-Miura ("Preparation of transforming deoxyribonucleic acid by phenoltreatment", extracted from Biochem. Biophys. Acta vol. 72, pp. 63-629, pp. 63-629).
[0151]
10 μg of the extracted DNA was completely digested with 10 U of restriction enzyme HindIII and randomly cloned into a vector pGEM-3Z (produced by PROMEGA). The cloned plasmid DNA was extracted according to the method described in Example 3- (1) below and transferred to a nylon filter. Southern blot hybridization was carried out using genomic DNA of Haemophilus influenzae standard strain genomic DNA labeled with digoxigenin-11-dUTP as a probe. That is, the nylon filter was subjected to hybridization under the conditions of 45% formamide, 5 × SSC and 42 ° C. overnight, and then the nylon filter was washed and colored according to the method described in Example 2. As a result, four types of DNA fragments that specifically reacted with Haemophilus influenzae genomic DNA were selected.
[0152]
In addition, each probe selected here was named probe HI-21-8, HI-57-34, HI-63-43, and HI-65-39.
[0153]
Example 2: Examination of species specificity of probe
The reactivity of each probe selected in Example 1 with DNA of various infectious disease causing strains was examined by the following method.
[0154]
First, consideration strains Hemofirisu influenza (Haemophilus influenzae clinical isolate), Hemofirisu parainfluenza (Haemophilus parainfluenzae Clinical isolates), Staphylococcus aureus (Staphylococcus aureus ATCC12600), Staphylococcus epidermidis (Staphylococcus epidermidis ATCC14990), Escherichia coli (Escherichia coli ATCC 11775), Klebsiella pneumoniae clinical isolate, Enterobacter cloacae clinical isolate, d. Terrorism Lactococcus Fe Car squirrel (Enterococcus faecalis ATCC19433), Citrobacter freundii (Citrobacter freundii clinical isolate) Bacteroides fragilis (Bacteroides fragilis SMUM-2275), Pseudomonas Jiminuta (Pseudomonas diminuta IFO14213), Micrococcus luteus (Micrococcus luteus JCM1464) and U937 ( Human genomic DNA) was prepared.
[0155]
Then, for each clinical strain, according to the method described in Example 1, DNA contained in each strain was extracted, and a fixed amount (for example, 10 to 100 ng) of the extracted DNA was spotted on a nylon filter, and alkali-denatured. These were used as dot blot hybridization samples. Then, using a probe labeled with Didigigenin-11-dUTP (manufactured by BRL), a manual of Maniatis (T. Maniatis, et al., Molecular Cloning (A Laboratory Manual Second Edition, Cold Spring Harbor, 89) is available. Hybridization was carried out overnight at 45% formamide, 5 × SSC and 42 ° C. The sample that had been subjected to the overnight hybridization was washed twice with 0.1 × SSC and 0.1% SDS at 55 ° C. for 20 minutes according to the manual, and then Anti-Dig-ALP conjugates (manufactured by BRL). And the color was developed to confirm the state of hybridization.
[0156]
As a result, it was confirmed that all of the probes HI-21-8, HI-57-34, HI-63-43 and HI-65-39 specifically reacted only with the genomic DNA of Haemophilus influenzae. Was.
[0157]
Example 3: Analysis of nucleotide sequence
The nucleotide sequences of the DNA probes (4 in total) whose species specificity was confirmed in Example 2 were determined according to the following method.
[0158]
(1) Plasmid DNA Preparation of
Escherichia coli K-12 and JM109 transformants containing the subcloned insert fragment (to be determined for nucleotide sequence) in pGEM-3Z (Promega) were added to 5 ml of Luria-Bactani Medium (bacto-tryptone, 10 g / 1; bacto-yeast extract, 5 g / l; NaCl, 10 g / l; adjusted to pH 7.0 with 5N NaOH) and cultured overnight. The culture was centrifuged (5,000 rpm, 5 minutes) and collected. 100 μl of a 50 mM glucose / 50 mM Tris-HCl (pH 8.0) / 10 mM EDTA solution containing lysozyme (Sigma) at a concentration of 2.5 mg / ml was added to the precipitate and left at room temperature for 5 minutes. A 0.2 M aqueous sodium hydroxide solution containing sodium dodecyl sulfate (Sigma) at a concentration of 1% was added to the obtained suspension and mixed.
[0159]
150 μl of a 5M aqueous potassium acetate solution (pH 4.8) was further added and mixed, followed by cooling on ice for 15 minutes. Then, the supernatant obtained by centrifugation (15,000 rpm, 15 minutes) was subjected to phenol / CHCl₃After the treatment, a 2-fold amount of ethanol was added to the supernatant, followed by centrifugation (12,000 rpm, 5 minutes) to obtain a precipitate. This precipitate was dissolved in 100 μl of a 10 mM Tris-HCl (pH 7.5) /0.1 mM EDTA solution, a 10 mg / ml RNaseA (Sigma) solution was added, and the mixture was left at room temperature for 15 minutes. To this preparation, 300 μl of a 0.1 M aqueous sodium acetate solution (pH 4.8) was added, and phenol / CHCl was added.₃After treatment, ethanol was added to the supernatant to obtain a precipitate. The precipitate was dried and dissolved in 10 μl of distilled water to obtain a DNA sample.
[0160]
(2) Preprocessing for nucleotide sequence determination
Preprocessing for base sequence determination was performed using AutoRead (registered trademark) Sequencing Kit (manufactured by Pharmacia).
[0161]
First, the DNA as a template was adjusted to a concentration of 5 to 10 μg in a 32 μl solution. 32 μl of the template DNA was transferred to a 1.5 ml mini tube (Eppendorf), and 8 μl of a 2 M aqueous sodium hydroxide solution was added thereto, followed by gentle mixing. After light centrifugation, the mixture was left at room temperature for 10 minutes. 7 μl of 3M sodium acetate (pH 4.8) and 4 μl of distilled water were added, and 120 μl of ethanol was further added, mixed, and left on ethanol-dry ice for 15 minutes. Then, the precipitated DNA was collected by centrifugation for 15 minutes, and the supernatant was carefully removed. The obtained precipitate was washed with 70% ethanol and centrifuged for 10 minutes. Then, the supernatant was again removed carefully, and the precipitate was dried under reduced pressure.
[0162]
The precipitate was dissolved in 10 μl of distilled water, and a fluorescent primer [Fluorescent Primer, Universal Primer; 5′-Fluorescein-d [CGACGTTGTAAAACGACGGCCAGT (SEQ ID NO: 5)]-3 ′, (1.6 pmol / μl; 0.42A)₂₆₀Unit / ml); Reverse Primer, 5'-Fluorescein-d [CAGGAAACAGCTATGAC (SEQ ID NO: 6)]-3 '(2.1 pmol / [mu] l; 0.42A)₂₆₀unit / ml)] 2 μl (0.42 A₂₆₀(unit / ml, 4-6 pmol) and 2 μl of annealing buffer were added and mixed gently. Then, after slightly centrifuging, heat treatment was performed at 65 ° C. for 5 minutes, and quickly placed under the condition of 37 ° C., where the temperature was maintained for 10 minutes. After the incubation, the mixture was left at room temperature for 10 minutes or more, and centrifuged lightly. Then, a sample to which 1 μl of the extension buffer and 3 μl of dimethyl sulfoxide were added was used as a sample.
[0163]
Mark A, C, G and T on the four minitubes, and in each tube Amix (ddATP dissolved with dATP, dCTP, c7dGTP and dTTP), C Mix (ddCTP with dATP, dCTP, c7dGTP and dTTP). ), G Mix (ddGTP dissolved with dATP, dCTP, c7dGTP and dTTP) and T Mix (ddTTP dissolved with dATP, dCTP, c7dGTP and dTTP) were dispensed in 2.5 μl aliquots. .
[0164]
Each solution was stored in water until use, and was kept warm at 37 ° C. for 1 minute or more before use. 2 μl of diluted T7 DNA polymerase (Pharmacia; 6-8 units / 2 μl) was added to the DNA sample and mixed thoroughly by pipetting or gentle mixing. Immediately after mixing, the mixed solution was dispensed into four kinds of solutions kept at 4.5 μl each. A new tip was used for dispensing.
[0165]
After incubation at 37 ° C. for 5 minutes, 5 μl of the stop solution was added to each reaction solution. Also in this dispensing, a new chip was used. The mixture was kept at 90 ° C for 2 to 3 minutes and immediately cooled in ice.
[0166]
Then, for electrophoresis, 4 to 6 μl of a sample was electrophoresed per lane.
[0167]
(3) Determination of nucleotide sequence
The determination of the base sequence of each probe having specificity for the DNA possessed by Haemophilus influenzae influenzae disclosed in Examples 1 and 2 was performed at a migration temperature of 45 ° C. and a migration time of 6 hours. L. F. This was performed using a DNA Sequencer system (Pharmacia). Primers were designed sequentially from the sequences revealed from each upstream and downstream, and the above operation was repeated.
[0168]
As a result, each base of the probes HI-21-8 (SEQ ID NO: 1), HI-57-34 (SEQ ID NO: 2), HI-63-43 (SEQ ID NO: 3) and HI-65-39 (SEQ ID NO: 4) The entire sequence was revealed.
[0169]
【The invention's effect】
As described above, according to the detection probe of the present invention, by applying it to in situ hybridization, it is possible to express a stable signal with respect to the bacteria to be detected in an extremely short time. It is possible to provide quick and accurate inspection results.
[0170]
In addition, this makes it possible to promptly provide medical materials with diagnostic materials for diseases such as sepsis and bacteremia as well as Haemophilus influenzae bacteria, and is expected to make a great contribution from the viewpoint of saving lives.
[0171]
[Sequence list]

Claims (17)

A probe for detecting Haemophilus influenzae, wherein the probe exhibits specific cross-reactivity with DNA carried by Haemophilus influenzae, and has the following nucleotide sequence:
(A) the nucleotide sequence of any one of SEQ ID NOs: 1 to 4;
(B) a base sequence having 70% or more homology with the base sequence (a); or
(C) a nucleotide sequence complementary to the nucleotide sequence (a) and / or (b),
A probe for detecting Haemophilus influenzae. The detection probe according to claim 1, wherein the base sequence is one or more nucleic acid fragments having a length of 350 to 600 bases. 3. The detection probe according to claim 1, wherein the DNA is chromosomal DNA. A method for detecting Haemophilus influenzae, comprising using the detection probe according to any one of claims 1 to 3. The detection method comprises the following steps:
(A) immobilizing a biologically-derived phagocyte obtained from a clinical sample on a support carrier,
(B) performing a chemical treatment to enhance the permeability of the cell membrane of the fixed phagocytes,
(C) obtaining chromosomal DNA of an infectious disease-causing bacterium contained in a phagocyte;
(D) performing in situ hybridization between the DNA and the detection probe according to any one of claims 1 to 3 under stringent conditions, and (e) detecting a hybridization signal.
The detection method according to claim 4, comprising a step. The clinical specimen is from the group consisting of blood, tissue fluid, lymph, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, ascites, dialysis drainage, tissue wash, skin, lung, kidney, mucosa, and combinations thereof. The detection method according to claim 5, which is selected. A method for identifying Haemophilus influenzae, comprising using the detection probe according to any one of claims 1 to 3. The identification method comprises the following steps:
(A) immobilizing a biologically-derived phagocyte obtained from a clinical sample on a support carrier,
(B) performing a chemical treatment to enhance the permeability of the cell membrane of the fixed phagocytes,
(C) obtaining chromosomal DNA of an infectious disease-causing bacterium contained in a phagocyte;
(D) subjecting the DNA to alkaline denaturation and neutralization treatment, and (e) performing dot blot hybridization between the DNA and the detection probe according to any one of claims 1 to 3 under stringent conditions. ,
The identification method according to claim 7, comprising a step. The clinical specimen is from the group consisting of blood, tissue fluid, lymph, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, ascites, dialysis drainage, tissue wash, skin, lung, kidney, mucosa, and combinations thereof. The identification method according to claim 8, which is selected. A method for identifying Haemophilus influenzae, comprising the following steps:
(A) obtaining chromosomal DNA of an infectious disease causing bacterium of unknown bacterial type,
(B) preparing a primer comprising at least a part of the base sequence constituting the detection probe according to any one of claims 1 to 3,
(C) amplifying the DNA by polymerase chain reaction (PCR) in a coexistence system of the DNA and the primer, and (d) detecting the amplified DNA;
A method for identifying Haemophilus influenzae, comprising the steps of: A method for observing the gene of Haemophilus influenzae phagocytosed by phagocytes, comprising the following steps:
(A) fixing phagocytes derived from a living body obtained from a clinical sample on a support,
(B) performing a chemical treatment to enhance the permeability of the cell membrane of the fixed phagocytes,
(C) obtaining chromosomal DNA of an infectious disease-causing bacterium contained in a phagocyte;
(D) performing in situ hybridization between the DNA and the detection probe according to any one of claims 1 to 3 under stringent conditions;
(E) detecting the hybridization signal,
(F) repeating steps (a)-(e), and (g) monitoring the change in hybridization signal over time;
A method for observing a gene of Haemophilus influenzae phagocytosed by a phagocyte, comprising the steps of: A kit for detecting Haemophilus influenzae, comprising the detection probe according to any one of claims 1 to 3 and a DNA exposure treatment agent. The detection kit according to claim 12, wherein the DNA exposure treatment agent includes an enzyme selected from the group consisting of lysostaphin, lysozyme, N-acetylmuramidase, zymolase, and a combination thereof. 14. The detection kit according to claim 12, wherein the detection probe coexists with a surfactant. The detection kit includes a blood separation reagent, an enzyme pretreatment reagent, an enzyme reagent, an acetylation reagent, a blocking reagent, a labeled antibody, a labeled antibody diluent, a color development pretreatment solution, a color development reagent, a counterstain solution, a PBS stock solution, and a hybridization stock solution. 15. The detection kit according to claim 12, further comprising a reagent selected from the group consisting of a labeled antibody washing solution, a coloring reagent washing solution, a probe diluting solution, a buffer A, and a combination thereof. The detection kit is an APS-coated slide glass, low-speed centrifuge, thermostat, hemocytometer, shaker, wet box, thermostat, optical microscope, variable pipette, blood collection tube, tip, pipette, staining bottle, measuring cylinder The detection kit according to any one of claims 12 to 15, further comprising an instrument selected from the group consisting of: a syringe, a syringe top filter, and a combination thereof. A DNA chip having a chip substrate and a DNA fragment having one end fixed to the surface of the substrate, wherein the fragment is the detection probe according to claim 1 or a fragment thereof. Characteristic DNA chip.