JP2020531024A - 微生物の特異的検出方法 - Google Patents
微生物の特異的検出方法 Download PDFInfo
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- JP2020531024A JP2020531024A JP2020511289A JP2020511289A JP2020531024A JP 2020531024 A JP2020531024 A JP 2020531024A JP 2020511289 A JP2020511289 A JP 2020511289A JP 2020511289 A JP2020511289 A JP 2020511289A JP 2020531024 A JP2020531024 A JP 2020531024A
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Abstract
Description
(a)試料を取得し;
(b)固定細胞を得るために、前記試料に含まれる細胞を固定剤で所定の位置に固定し、それによって得られた前記固定細胞を前記試料から分離し;
(c)乾燥細胞を得るために、前記固定細胞を化学均質剤に接触させ、それによって得られた均質化された細胞を乾燥させ;
(d)第1反応混合物を得るために、前記乾燥細胞を検出対象の微生物に対して特異的な蛍光標識核酸プローブの溶液に接触させ;
(e)前記検出対象の微生物の細胞内の対応する標的核酸配列に前記蛍光標識核酸プローブを結合させるために、前記第1反応混合物をインキュベートし;
(f)第2反応混合物を得るために、工程(e)に続いて、前記第1反応混合物を消光標識核酸プローブの溶液に接触させ(ここで、前記消光標識核酸プローブは、前記蛍光標識核酸プローブの蛍光を少なくとも部分的に消光する消光剤を含み、前記蛍光標識核酸プローブの核酸配列に略相補的な核酸配列を含む);
(g)前記検出対象の微生物の細胞内の標的核酸配列に結合していない前記蛍光標識核酸プローブの分子の前記消光標識核酸プローブとの結合を引き起こすために、前記第2反応混合物をインキュベートし、;
(h)工程(g)後に、前記蛍光標識核酸プローブを含む前記検出対象の微生物の細胞から放出される蛍光を検出するために、前記第2反応混合物をフロースルーサイトメータに配置する。
微生物の特異的検出方法としては、果汁飲料中のアリシクロバチルス種の検出を例示する。
固定細胞を乾燥させる前に均質剤を添加しないことを除いて、上述した実施例1を繰り返した。その結果を図3に示す。
上記の実施例1は、国際公開公報WO 03/083131 A1に記載の方法を採用し、消光標識核酸プローブを使用しなかったことを除いて、実質的に繰り返される。その結果を図4に示す。
実施例1で使用した均質剤の代わりに、70%エタノールを均質剤として使用したことを除いて、上記実施例1を繰り返した。その結果を図5に示す。
Claims (15)
- (a)試料を取得し;
(b)固定細胞を得るために、前記試料に含まれる細胞を固定剤で所定の位置に固定し、それによって得られた前記固定細胞を前記試料から分離し;
(c)乾燥細胞を得るために、前記固定細胞を化学均質剤に接触させ、それによって得られた均質化された細胞を乾燥させ;
(d)第1反応混合物を得るために、前記乾燥細胞を検出対象の微生物に対して特異的な蛍光標識核酸プローブの溶液に接触させ;
(e)前記検出対象の微生物の細胞内の対応する標的核酸配列に前記蛍光標識核酸プローブを結合させるために、前記第1反応混合物をインキュベートし;
(f)第2反応混合物を得るために、工程(e)に続いて、前記第1反応混合物を消光標識核酸プローブの溶液に接触させ(ここで、前記消光標識核酸プローブは、前記蛍光標識核酸プローブの蛍光を少なくとも部分的に消光する消光剤を含み、前記蛍光標識核酸プローブの核酸配列に略相補的な核酸配列を含む);
(g)前記検出対象の微生物の細胞内の標的核酸配列に結合していない前記蛍光標識核酸プローブの分子の前記消光標識核酸プローブとの結合を引き起こすために、前記第2反応混合物をインキュベートし;
(h)工程(g)後に、前記蛍光標識核酸プローブを含む前記検出対象の微生物の細胞から放出される蛍光を検出するために、前記第2反応混合物をフロースルーサイトメータに配置する、工程を有することを特徴とする試料中微生物の検出方法。 - 前記試料は液体試料であることを特徴とする請求項1に記載の方法。
- 前記微生物は細菌、真菌、又は単細胞上位生物(原生動物)であることを特徴とする請求項1又は2に記載の方法。
- 前記細菌は、アシネトバクター属、アリシクロバチルス属、アクアバクテリア属、アルコバクター属、バチルス属、カンピロバクター属、腸内細菌科、エシェリヒア属、ラクトバチルス属、ラクトコッカス属、レジオネラ属、リステリア属、マイクロトリックス属、ニトロバクター属、ニトロソコッカス属、ニトロソモナス属、ニトロスピラ属、ニトロトガ属、プロピオニバクテリウム属、サルモネラ属、志賀菌属、又は連鎖球菌属からの細菌であることを特徴とする請求項3に記載の方法。
- 前記真菌は、アスペルギルス属、カンジダ属、デバロマイセス属、デッケラ属、ペニシリウム属、ピチア属、又はサッカロミセス属からの真菌であることを特徴とする請求項3に記載の方法。
- 前記均質剤は、(a)単糖類又は二糖類、(b)ポリオール、及び(c)水を含むことを特徴とする請求項1〜5いずれか一項に記載の方法。
- 前記単糖類又は二糖類が、フルクトース、ガラクトース、グルコース及びスクロースからなる群から選択される物質であることを特徴とする請求項6に記載の方法。
- 前記ポリオールが、エチレングリコール、グリセリン、マンニトール及びソルビトールからなる群から選択される物質であることを特徴とする請求項6又は7に記載の方法。
- 前記検出対象の前記微生物の細胞標的核酸配列は、16S rRNA、23S rRNA、18S rRNA、tRNA、EF−Tu、mRNA 16S−23S rRNAスペーサー、及び23S−5S rRNAスペーサーからなる群から選択されることを特徴とする請求項1〜8のいずれか一項に記載の方法。
- 前記蛍光標識核酸プローブは、前記検出対象の微生物の細胞中の標的核酸配列と(i)略同一であること、又は(ii)略逆相補的であることを特徴とする請求項1〜9のいずれか一項に記載の方法。
- 前記蛍光標識核酸プローブは、蛍光標識DNAプローブ、RNAプローブ、PNAプローブ、及びLNAプローブから選択されることを特徴とする請求項1〜10のいずれか一項に記載の方法。
- (i)前記蛍光標識核酸プローブの蛍光色素は、前記蛍光標識核酸プローブの3’末端若しくは又は3’末端近くに位置し、そして前記消光標識核酸プローブの消光剤は、前記消光標識核酸プローブの5’末端若しくは5’末端近くに位置し、又は、(ii)前記蛍光標識核酸プローブの蛍光色素は、前記蛍光標識核酸プローブの5’末端若しくは5’末端近くに位置し、そして前記消光標識核酸プローブの消光剤は、前記消光標識核酸プローブの3’末端若しくは3’末端近くに位置することを特徴とする請求項1〜11のいずれか一項に記載の方法。
- 工程(d)において、検出対象の各微生物に対して特異的な異なる核酸配列を有する多数の蛍光標識核酸プローブを添加し、そして、工程(f)において、蛍光標識核酸プローブの数に対応する多数の異なる消光標識核酸プローブを添加することを特徴とする請求項1〜12のいずれか一項に記載の方法。
- 試料は1種より多くの微生物を含み、そして多数の異なる微生物は同時に検出されることを特徴とする請求項1〜13のいずれか一項に記載の方法。
- 請求項1〜14のいずれか一項に記載の方法で使用するためのフロースルーサイトメータ。
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