JP2022533269A - 糞便試料中の希少なdna配列の検出方法 - Google Patents
糞便試料中の希少なdna配列の検出方法 Download PDFInfo
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Abstract
Description
本出願は2019年5月23日に出願された米国特許出願第62/852,018号の恩典を主張するものであり、その内容は参照により本明細書に組み込まれる。
本開示は、概して、糞便試料中の希少DNA配列を検出するための方法および組成物を提供する。
本出願は、ASCII形式で電子的に提出された配列表を含み、その全体は参照により本明細書に組み込まれる。2020年5月26日に作成された当該ASCIIコピーは、「116110-5005-WO_ST25_Sequence_Listing」と名付けられ、サイズは8キロバイトである。
糞便は容易に入手でき、代謝廃棄物の豊富な供給源である。糞便は、腸管内腔と血液循環との間を遊走するマクロファージおよびリンパ球を含む数百万の宿主細胞と共に、哺乳類の消化管内に常駐する数兆もの微生物を含有する。ヒト腸内微生物叢に関して増えつつある知識は、消化管内の細菌分類群の組成が、神経学、精神医学、呼吸、心血管、胃腸、肝臓、自己免疫、代謝および腫瘍学などの領域の多数の障害を伴う、ホメオスタシスおよび疾患にとって重要であることを示す。さらに、糞便中に存在する異常な宿主細胞は、疾患の遺伝的シグネチャを有する。したがって、糞便は、病原体検出、腸内微生物叢分析、および疾患診断のための貴重かつ非侵襲的な生体材料の供給源を提供し、診断、疾患予測、および治療的介入において貴重な応用の可能性を秘めている。
本開示は、例えば、病原性細菌種からの低コピー数DNA配列を含む、糞便試料中の低コピー数DNA配列を同定するための方法および物質に関する。一部の実施形態では、本開示は、糞便試料中のがんなどの疾患に関連する低コピー数遺伝的変異体の同定に関する。さらなる実施形態では、本開示は、定量的手段もしくは半定量的手段による検出のために、または配列決定による検出のために、低コピー数DNA配列を濃縮する方法に関する。本開示はまた、糞便試料からの低コピー数DNA配列を含む配列決定ライブラリを調製するための方法および組成物に関する。さらに、本開示は、標識されたオリゴヌクレオチドを使用して試料中の十分な細菌種を枯渇させるための組成物およびキットに関する。
本開示は、例えば、病原性細菌種からの低コピー数DNA配列を含む、糞便試料中の低コピー数DNA配列を同定するための方法および物質に関する。一部の実施形態では、本開示は、糞便試料中のがんなどの疾患に関連する低コピー数遺伝的変異体の同定に関する。
本明細書に開示される方法の実施形態に従った低コピー数DNA配列の検出の実現可能性を決定するために、NGSに基づく配列検出アッセイを実施した。ピロリ菌株26695のゲノムDNAをATCCから購入した。ピロリ菌抗生物質耐性に関連する6つの遺伝子の突然変異を検出するための次世代配列決定の感受性を試験するために、26695ゲノムDNAを、100万コピーから0.1コピーまでの一連のコピーに希釈し、次いでライブラリ調製物に使用した。各コピー数の希釈を3回実施したが、100万コピーおよび10万コピーの試料は例外で、2回ずつ実施した。得られたライブラリを、Illumina(登録商標)MiSeq(登録商標)プラットフォームを使用して配列決定した。
サルモネラに感染(Luminexにより検出)した糞便試料と、対照細菌(カンピロバクター菌(Campylobacter)、サルモネラ菌(Salmonella)、赤痢菌(Shigella)、ビブリオ菌(Vibrio)、エルシニア・エンテロコリチカ菌(Yersinia enterolitica)、および志賀毒素産生大腸菌)を接種した緩衝液を含む対照から、総ゲノムDNAを抽出した。抗生物質耐性株を含む、微生物群における細菌、ウイルス、真菌、原生生物などを検出するために、分析したすべての配列の相対存在量情報を評価するため、試料中のすべてのDNA物質を標的とするショットガンメタゲノミクス法を実施した。ショットガンメタゲノミクス法は、ゲノムの塩基対レベルの分解能を提供し、抗生物質耐性解析および株同定のための単一のヌクレオチド変異体解析を可能にする。
Claims (23)
- 糞便試料中の低コピー数DNA配列を同定する方法であって、
対象から糞便試料を取得する工程と、
DNA調製物を得るために前記糞便試料からDNAを抽出する工程と、
ハイブリダイゼーション複合体を形成するために、前記DNA調製物において、標識されたオリゴヌクレオチドプローブを非病原性細菌DNA配列にハイブリダイズする工程と、
前記DNA調製物から前記ハイブリダイゼーション複合体を枯渇させる工程と、
前記DNA調製物中の前記低コピー数DNA配列の存在を同定する工程と
を含み、
前記DNA調製物中の前記低コピー数DNA配列の同定が、前記低コピー数DNA配列が前記糞便試料中に存在することを示す、方法。 - 前記低コピー数DNA配列が、ピロリ菌(H. pylori)DNA配列である、請求項1に記載の方法。
- 前記低コピー数DNA配列が、ヒトDNA配列である、請求項1に記載の方法。
- 前記ヒトDNA配列が、がん状態または前がん状態と関連している、請求項3に記載の方法。
- 前記非病原性細菌DNAが、バクテロイデス属(Bacteroides)、クロストリジウム属(Clostridium)、フィーカリバクテリウム属(Faecalibacterium)、またはそれらの組み合わせに由来する、請求項1に記載の方法。
- 前記標識されたオリゴヌクレオチドプローブが、前記非病原性細菌DNAの保存領域に相補的である、請求項1に記載の方法。
- 前記標識がビオチンである、請求項1に記載の方法。
- 前記ビオチン標識されたハイブリダイゼーション複合体をストレプトアビジン被覆基体と共にインキュベートする工程をさらに含む、請求項7に記載の方法。
- 前記ストレプトアビジン被覆基体が、ビーズ、カラム、または膜を含む、請求項8に記載の方法。
- 前記ストレプトアビジン被覆基体が、磁気ビーズを含み、前記ハイブリダイゼーション複合体が、磁界を使用して前記DNA調製物から枯渇される、請求項8に記載の方法。
- 前記ハイブリダイゼーション複合体が、遠心力を使用して前記DNA調製物から枯渇される、請求項1に記載の方法。
- 前記非病原性細菌DNAが、バクテロイデス・フラジリス(Bacteroides fragilis)、バクテロイデス・メラニノゲニクス(Bacteroides melaninogenicus)、バクテロイデス・オラリス(Bacteroides oralis)、またはそれらの組み合わせに由来する、請求項1に記載の方法。
- 前記標識されたオリゴヌクレオチドプローブが、配列番号1、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、または配列番号8から選択される、請求項12に記載の方法。
- 低コピー数DNA配列を同定する工程が、前記DNA配列を配列決定することをさらに含む、請求項1に記載の方法。
- 低コピー数DNA配列を同定する工程が、定量PCR反応を含む、請求項1に記載の方法。
- 低コピー数DNA配列を同定する工程が、前記試料を一つ以上の追加試料と多重化することを含む、請求項1に記載の方法。
- 前記糞便試料から抽出されたDNAが、約0.5グラム~約1.0グラムの重量である、請求項1に記載の方法。
- 総DNAが前記糞便試料から抽出される、請求項1に記載の方法。
- 前記DNAを抽出する工程が、溶解緩衝液中で前記糞便試料をビーズ粉砕することを含み、前記溶解緩衝液が、細菌細胞壁を破壊すること、タンパク質を消化すること、タンパク質を変性すること、脂肪を分散すること、多糖類を沈殿させること、またはそれらの組み合わせが可能な成分を含む、請求項1に記載の方法。
- 糞便試料中の低コピー数DNA配列を濃縮する方法であって、
対象から糞便試料を取得する工程と、
DNA調製物を得るために前記糞便試料からDNAを抽出する工程と、
ハイブリダイゼーション複合体を形成するために、前記DNA調製物において、標識されたオリゴヌクレオチドプローブを非病原性細菌DNA配列にハイブリダイズする工程と、
前記DNA調製物から前記ハイブリダイゼーション複合体を枯渇させる工程と
を含む、方法。 - 糞便試料中の抗生物質耐性ピロリ菌を同定する方法であって、
対象から糞便試料を取得する工程と、
DNA調製物を得るために前記糞便試料からDNAを抽出する工程と、
ハイブリダイゼーション複合体を形成するために、前記DNA調製物において、標識されたオリゴヌクレオチドプローブを非病原性細菌DNA配列にハイブリダイズする工程と、
前記DNA調製物から前記ハイブリダイゼーション複合体を枯渇させる工程と、
前記DNA調製物中のピロリ菌DNAの領域の多重コピーを生成するために、前記ピロリ菌DNAの前記領域を増幅する工程と、
前記ピロリ菌DNAの前記増幅領域の前記多重コピーを配列決定する工程と、
前記ピロリ菌DNAの前記増幅領域の前記多重コピーの配列を、参照配列と比較する工程と、
前記ピロリ菌DNAの前記領域の前記多重コピーにおける突然変異の存在を同定する工程と、
前記突然変異を有する前記ピロリ菌DNAの前記領域の前記多重コピーの数を決定する工程と
を含み、
前記突然変異を有する前記ピロリ菌DNAの前記領域の前記多重コピーの前記数が所定量を超えるときに、抗生物質耐性ピロリ菌が前記試料中に存在する、方法。 - 糞便試料中の低コピー数DNA配列を含む次世代配列決定ライブラリを調製する方法であって、
対象から糞便試料を取得する工程と、
DNA調製物を得るために前記糞便試料からDNAを抽出する工程と、
ハイブリダイゼーション複合体を形成するために、前記DNA調製物において、標識されたオリゴヌクレオチドプローブを非病原性細菌DNA配列にハイブリダイズする工程と、
前記DNA調製物から前記ハイブリダイゼーション複合体を枯渇させる工程と、
前記枯渇させたDNA調製物において一つ以上の単位複製配列を増幅する工程と
を含む、方法。 - ヒト対象においてがんを検出する方法であって、
対象から糞便試料を取得する工程と、
DNA調製物を得るために前記糞便試料からDNAを抽出する工程と、
ハイブリダイゼーション複合体を形成するために、前記DNA調製物において、標識されたオリゴヌクレオチドプローブを非病原性細菌DNA配列にハイブリダイズする工程と、
前記DNA調製物から前記ハイブリダイゼーション複合体を枯渇させる工程と、
前記試料中の一つ以上の希少ながん関連DNA配列の存在を検出する工程と
を含む、方法。
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