JP6979409B2 - 細胞溶解及び核酸増幅のためのプロセス - Google Patents
細胞溶解及び核酸増幅のためのプロセス Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
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Description
本出願は、2016年4月8日に出願された米国仮特許出願第62/319,876号の優先権を主張するものであり、この開示内容は、その全容が参照により本明細書に組み込まれる。
実施形態Aは核酸増幅法であって、
試料を、水溶性リン酸イオンを実質的に含まず第1の緩衝液を含む栄養培地と接触させることによって、集積培養物を形成することと、
集積培養物を、ある時間、標的微生物の生育を促進する温度に維持することと、
集積培養物を維持した後に、第1の体積の集積培養物を第2の体積の溶解緩衝液と混合し、第3の体積の水性組成物を形成することにより、水性組成物を形成することであって、
第1の体積の集積培養物を第2の体積の溶解緩衝液と混合することが、無希釈の第1の体積の集積培養物を第2の体積の溶解緩衝液と混合することを含み、
溶解緩衝液が水溶性リン酸イオンを実質的に含まず、第2の緩衝液を含み、
溶解緩衝液が有機多価陽イオンキレート試薬を含み、有機多価陽イオンキレート試薬が、三価鉄に対して104.2以上の第1の結合定数と、マグネシウムに対して103.8未満の第2の結合定数とを有し、第1の結合定数及び第2の結合定数が、20℃の脱イオン水中、pH8.45で測定されたものであり、
溶解緩衝液は、25℃でpHが8.6超であり、
水性組成物は、25℃でpHが約8.45〜8.85であり、
水性組成物中の第1の緩衝液と第2の緩衝液との合算した濃度が、少なくとも約15mMであり、
第1の体積の第2の体積に対する比が3:1以上であり、第2の体積の第3の体積に対する比が1:4以下である、水性組成物を形成することと、
水性組成物を、ポリメラーゼ媒介核酸増幅反応を阻害する物質を封鎖する有効量の水不溶性材料と接触させることと、
水性組成物を熱溶解プロセスに付すことと、
水性組成物を熱溶解プロセスに付した後に、水性組成物の一部分を核酸増幅プロセスに付すことと、
を含む、方法である。
試料を、リン酸緩衝液構成成分を含まない配合を有し、第1の緩衝液を含む栄養培地と接触させることによって、集積培養物を形成することと、
集積培養物を、ある時間、標的微生物の生育を促進する温度に維持することと、
集積培養物を維持した後に、第1の体積の集積培養物を第2の体積の溶解緩衝液と混合し、第3の体積の水性組成物を形成することにより、水性組成物を形成することであって、
第1の体積の集積培養物を第2の体積の溶解緩衝液と混合することが、無希釈の第1の体積の集積培養物を第2の体積の溶解緩衝液と混合することを含み、
溶解緩衝液が第2の緩衝液を含み、
溶解緩衝液が有機多価陽イオンキレート試薬を含み、有機多価陽イオンキレート試薬が、三価鉄に対して104.2以上の第1の結合定数と、マグネシウムに対して103.8未満の第2の結合定数とを有し、第1の結合定数及び第2の結合定数が、20℃の脱イオン水中、pH8.45で測定されたものであり、
溶解緩衝液は、25℃でpHが8.6超であり、
水性組成物は、25℃でpHが約8.45〜8.85であり、
水性組成物中の第1の緩衝液と第2の緩衝液との合算した濃度が、少なくとも約15mMであり、
第1の体積の第2の体積に対する比が3:1以上であり、第2の体積の第3の体積に対する比が1:4以下である、水性組成物を形成することと、
水性組成物を、ポリメラーゼ媒介核酸増幅反応を阻害する物質を封鎖する有効量の水不溶性材料と接触させることと、
水性組成物を熱溶解プロセスに付すことと、
水性組成物を熱溶解プロセスに付した後に、水性組成物の一部分を核酸増幅プロセスに付すことと、
を含む、方法である。
水性組成物を熱溶解プロセスに付した後、かつ水性組成物の一部分を核酸増幅プロセスに付す前に、水性組成物の一部分を用い、核酸増幅用の脱水された試薬を再水和すること、を更に含む、請求項A〜Qのいずれか1つに記載の方法である。
ポリメラーゼ媒介核酸増幅反応を阻害する物質を封鎖する水不溶性材料と、
有機多価陽イオンキレート試薬と、
少なくとも1種のノニオン性界面活性剤と、
三価鉄と、
ポリメラーゼ活性を促進する試薬と、
緩衝液と、
水と、
を含み、
1mM未満のリン酸又はその塩を含み、
25℃でpHが約9.8〜10.5(両端の値を含む)である、溶解緩衝液である。
Claims (5)
- 核酸増幅法であって、
試料を、リン酸緩衝液構成成分を含まない配合を有し、第1の緩衝液を含む栄養培地と接触させることによって、集積培養物を形成することと、
前記集積培養物を、ある時間、標的微生物の生育を促進する温度に維持することと、
前記集積培養物を維持した後に、第1の体積の前記集積培養物を第2の体積の溶解緩衝液と混合し、第3の体積の水性組成物を形成することにより、前記水性組成物を形成することであって、
前記第1の体積の前記集積培養物を前記第2の体積の前記溶解緩衝液と混合することが、無希釈の第1の体積の前記集積培養物を前記第2の体積の前記溶解緩衝液と混合することを含み、
前記溶解緩衝液が第2の緩衝液を含み、
前記溶解緩衝液が有機多価陽イオンキレート試薬を含み、前記有機多価陽イオンキレート試薬が、エチレングリコール−ビス(2−アミノエチルエーテル)−N,N,N’,N’−四酢酸(EGTA)、N,N,N’,N’−テトラキス(2−ピリジルメチル)エタン−1,2−ジアミン(TPEN)、1,2−ビス(o−アミノフェノキシ)エタン−N,N,N’,N’−四酢酸(BAPTA)、N−(2−ヒドロキシエチル)エチレンジアミン−N,N’,N’−三酢酸(HEDTA)、及びこれらの塩から選択されたものであり、
前記溶解緩衝液は、25℃でpHが8.6超であり、
前記水性組成物は、25℃でpHが約8.45〜8.85であり、
前記水性組成物中の前記第1の緩衝液と前記第2の緩衝液との合算した濃度が、少なくとも約15mMであり、
前記第1の体積の前記第2の体積に対する比が3:1以上であり、前記第2の体積の前記第3の体積に対する比が1:4以下である、前記水性組成物を形成することと、
前記水性組成物を、ポリメラーゼ媒介核酸増幅反応を阻害する物質を封鎖する有効量の水不溶性材料と接触させることであって、
前記水不溶性材料が、酸化ジルコニウム粒子、ヒドロキシアパタイト粒子、及びこれらの混合物から選択される、前記水性組成物を前記水不溶性材料と接触させることと、
前記水性組成物を熱溶解プロセスに付すことと、
前記水性組成物を前記熱溶解プロセスに付した後に、前記水性組成物の一部分を核酸増幅プロセスに付すことと、
を含む、方法。 - 前記溶解緩衝液が三価鉄を更に含む、請求項1に記載の方法。
- 前記第1の体積の前記第2の体積に対する前記比が5:1以上であり、前記第2の体積の前記第3の体積に対する前記比が1:6以下である、請求項1又は2に記載の方法。
- 前記水不溶性材料が、前記第3の体積中、約10m2/L〜約600m2/Lの表面積を有する、複数の酸化ジルコニウム粒子を含む、請求項1〜3のいずれか一項に記載の方法。
- 前記水性組成物を前記熱溶解プロセスに付した後、かつ前記水性組成物の前記一部分を前記核酸増幅プロセスに付す前に、前記水性組成物の前記一部分を用い、核酸増幅用の脱水された試薬を再水和すること、を更に含む、請求項1〜4のいずれか一項に記載の方法。
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