JPWO2021034741A5

JPWO2021034741A5 -

Info

Publication number: JPWO2021034741A5
Application number: JP2022510896A
Authority: JP
Publication date: 2023-08-25

Description

[本発明1001]
生体試料から増幅可能な核酸を得る方法であって、
(a) 該生体試料を過炭酸塩、ヌクレアーゼ抑制剤、およびキレート剤と接触させて、酸化還元反応組成物を形成させる段階；
(b) 該酸化還元反応組成物を、20～65℃である第1温度でインキュベートする段階；ならびに
(c) 該酸化還元反応組成物を、60～100℃である第2温度でインキュベートする段階
を含む、前記方法。
[本発明1002]
前記酸化還元反応組成物が、前記第1温度で1～3分間インキュベートされる、本発明1001の方法。
[本発明1003]
前記酸化還元反応組成物が、前記第2温度で30～90秒間インキュベートされる、本発明1001または1002の方法。
[本発明1004]
前記酸化還元反応組成物を撹拌する段階をさらに含む、本発明1001～1003のいずれかの方法。
[本発明1005]
前記酸化還元反応組成物が15～90秒間撹拌される、本発明1004の方法。
[本発明1006]
前記酸化還元反応組成物が、(i) 前記第1温度でインキュベートする段階中、(ii) 前記第1温度でインキュベートする段階と前記第2温度でインキュベートする段階との間；(iii) 前記第2温度でインキュベートする段階中；および／または (iv) 前記第2温度でインキュベートする段階の後のうちのいずれか1つまたは複数において撹拌される、本発明1004の方法。
[本発明1007]
前記酸化還元反応組成物が、(i) 前記第1温度でインキュベートする段階と前記第2温度でインキュベートする段階との間、および (ii) 前記第2温度でインキュベートする段階の後に撹拌される、本発明1006の方法。
[本発明1008]
前記酸化還元反応組成物が、前記第1温度でインキュベートする段階と前記第2温度でインキュベートする段階との間に15～90秒間、および前記第2温度でインキュベートする段階の後に15～90秒間撹拌される、本発明1007の方法。
[本発明1009]
前記第1温度が35～60℃である、本発明1001～1008のいずれかの方法。
[本発明1010]
前記第2温度が70～95℃である、本発明1001～1009のいずれかの方法。
[本発明1011]
前記撹拌が、機械的撹拌または超音波処理を含む、本発明1001～1010のいずれかの方法。
[本発明1012]
前記生体試料をビーズと接触させる段階をさらに含む、本発明1001～1011のいずれかの方法。
[本発明1013]
前記ビーズが、シリカビーズまたはガラスビーズである、本発明1012の方法。
[本発明1014]
前記過炭酸塩が過炭酸ナトリウムを含む、本発明1001～1013のいずれかの方法。
[本発明1015]
前記ヌクレアーゼ抑制剤がプロテイナーゼKを含む、本発明1001～1014のいずれかの方法。
[本発明1016]
前記キレート剤がエチレンジアミン四酢酸 (EDTA) を含む、本発明1001～1015のいずれかの方法。
[本発明1017]
前記生体試料が、前記酸化還元反応組成物の形成前に緩衝液で希釈される、本発明1001～1016のいずれかの方法。
[本発明1018]
前記生体試料が、前記酸化還元反応組成物の形成前に緩衝液で1:1～1:5に希釈される、本発明1001～1017のいずれかの方法。
[本発明1019]
前記緩衝液がTrisである、本発明1018の方法。
[本発明1020]
前記増幅可能な核酸の少なくとも一部を増幅する段階をさらに含む、本発明1001～1019のいずれかの方法。
[本発明1021]
前記増幅可能な核酸の少なくとも一部を増幅する段階が、PCRまたはRT-PCRを実施することを含む、本発明1020の方法。
[本発明1022]
前記増幅可能な核酸の少なくとも一部を増幅する前に、前記酸化還元反応組成物に炭酸水素ナトリウムもチオ硫酸ナトリウムも添加されない、本発明1020または1021の方法。
[本発明1023]
前記増幅可能な核酸の少なくとも一部を増幅する前に、前記酸化還元反応組成物に対して洗浄段階が実施されない、本発明1020～1022のいずれかの方法。
[本発明1024]
前記酸化還元反応組成物が、前記増幅可能な核酸の少なくとも一部をPCRまたはRT-PCRによって増幅する前に緩衝液で希釈される、本発明1020～1023のいずれかの方法。
[本発明1025]
前記酸化還元反応組成物が、前記増幅可能な核酸の少なくとも一部をPCRまたはRT-PCRによって増幅する前に緩衝液で1:1～1:5に希釈される、本発明1020～1024のいずれかの方法。
[本発明1026]
過炭酸塩、ヌクレアーゼ抑制剤、およびキレート剤を含み、乾燥形態である、組成物。
[本発明1027]
前記過炭酸塩が過炭酸ナトリウムを含む、本発明1026の組成物。
[本発明1028]
前記ヌクレアーゼ抑制剤がプロテイナーゼKを含む、本発明1026または1027の組成物。
[本発明1029]
前記キレート剤がエチレンジアミン四酢酸 (EDTA) を含む、本発明1026～1028のいずれかの組成物。
[本発明1030]
ビーズをさらに含む、本発明1026～1029のいずれかの組成物。
[本発明1031]
アスコルビン酸を本質的に含まない、本発明1026～1030のいずれかの組成物。
本発明の他の目的、特徴、および利点は、以下の詳細な説明から明らかになるであろう。しかしながら、本発明の精神および範囲の範囲内の様々な変更および修正が、この詳細な説明から当業者に明らかになると考えられるため、詳細な説明および具体例は、本発明の好ましい態様を示しながら、例示として与えられているにすぎないことが理解されるべきである。 [Invention 1001]
A method of obtaining an amplifiable nucleic acid from a biological sample, comprising:
(a) contacting the biological sample with a percarbonate, a nuclease inhibitor, and a chelating agent to form a redox reaction composition;
(b) incubating the redox reaction composition at a first temperature that is between 20 and 65°C; and
(c) incubating the redox reaction composition at a second temperature that is between 60 and 100°C;
The above method, comprising
[Invention 1002]
1002. The method of invention 1001, wherein said redox reaction composition is incubated at said first temperature for 1-3 minutes.
[Invention 1003]
The method of invention 1001 or 1002, wherein said redox reaction composition is incubated at said second temperature for 30-90 seconds.
[Invention 1004]
The method of any of inventions 1001-1003, further comprising agitating said redox reaction composition.
[Invention 1005]
1004. The method of the present invention 1004, wherein said redox reactive composition is agitated for 15-90 seconds.
[Invention 1006]
(ii) between incubating at the first temperature and at the second temperature; (iii) during the step of incubating at the second temperature; and/or (iv) after incubating at said second temperature.
[Invention 1007]
the redox reaction composition is agitated (i) between the steps of incubating at the first temperature and the step of incubating at the second temperature, and (ii) after the step of incubating at the second temperature; The method of the invention 1006.
[Invention 1008]
The redox reaction composition is agitated for 15-90 seconds between incubating at the first temperature and at the second temperature, and for 15-90 seconds after incubating at the second temperature. The method of the invention 1007, wherein:
[Invention 1009]
1009. The method of any of the inventions 1001-1008, wherein said first temperature is 35-60°C.
[Invention 1010]
1009. The method of any of inventions 1001-1009, wherein said second temperature is 70-95°C.
[Invention 1011]
1010. The method of any of Inventions 1001-1010, wherein said agitation comprises mechanical agitation or sonication.
[Invention 1012]
The method of any of inventions 1001-1011, further comprising contacting said biological sample with beads.
[Invention 1013]
1013. The method of the present invention 1012, wherein said beads are silica beads or glass beads.
[Invention 1014]
1013. The method of any of inventions 1001-1013, wherein said percarbonate comprises sodium percarbonate.
[Invention 1015]
1014. The method of any of inventions 1001-1014, wherein said nuclease inhibitor comprises proteinase K.
[Invention 1016]
1015. The method of any of claims 1001-1015, wherein said chelating agent comprises ethylenediaminetetraacetic acid (EDTA).
[Invention 1017]
The method of any of inventions 1001-1016, wherein said biological sample is diluted with a buffer prior to formation of said redox reaction composition.
[Invention 1018]
1018. The method of any of inventions 1001-1017, wherein the biological sample is diluted 1:1 to 1:5 with a buffer prior to formation of the redox reaction composition.
[Invention 1019]
1018. The method of the invention 1018, wherein said buffer is Tris.
[Invention 1020]
The method of any of inventions 1001-1019, further comprising amplifying at least a portion of said amplifiable nucleic acid.
[Invention 1021]
The method of invention 1020, wherein amplifying at least a portion of said amplifiable nucleic acid comprises performing PCR or RT-PCR.
[Invention 1022]
The method of invention 1020 or 1021, wherein neither sodium bicarbonate nor sodium thiosulfate is added to said redox reaction composition prior to amplifying at least a portion of said amplifiable nucleic acid.
[Invention 1023]
The method of any of inventions 1020-1022, wherein no washing step is performed on said redox reaction composition prior to amplifying at least a portion of said amplifiable nucleic acid.
[Invention 1024]
1024. The method of any of inventions 1020-1023, wherein said redox reaction composition is diluted with a buffer prior to amplifying at least a portion of said amplifiable nucleic acid by PCR or RT-PCR.
[Invention 1025]
1024. Any of the inventions 1020-1024, wherein said redox reaction composition is diluted 1:1 to 1:5 with a buffer prior to amplifying at least a portion of said amplifiable nucleic acid by PCR or RT-PCR. method.
[Invention 1026]
A composition comprising a percarbonate, a nuclease inhibitor, and a chelating agent, wherein the composition is in dry form.
[Invention 1027]
1026. The composition of invention 1026, wherein said percarbonate comprises sodium percarbonate.
[Invention 1028]
The composition of invention 1026 or 1027, wherein said nuclease inhibitor comprises proteinase K.
[Invention 1029]
The composition of any of inventions 1026-1028, wherein said chelating agent comprises ethylenediaminetetraacetic acid (EDTA).
[Invention 1030]
The composition of any of inventions 1026-1029, further comprising beads.
[Invention 1031]
The composition of any of Inventions 1026-1030, which is essentially free of ascorbic acid.
Other objects, features, and advantages of the present invention will become apparent from the detailed description below. It is intended, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, will become apparent to those skilled in the art from this detailed description, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. , are given as examples only.

Claims

A method of obtaining an amplifiable nucleic acid from a biological sample, comprising:
(a) contacting the biological sample with a percarbonate, a nuclease inhibitor, and a chelating agent to form a redox reactant ;
(b) incubating the redox reaction at 20-65° C. for at least 1 minute ;
( c) incubating the redox reaction at 60-100° C. for at least 30 seconds ; and
(d) stirring the redox reactant;
The above method, comprising

2. The method of claim 1 , wherein step (b) comprises incubating the redox reaction at 20-65°C for at least 2 minutes .

3. The method of claim 1 or 2, wherein step (b) comprises incubating the redox reaction at 60<0>C for at least 2 minutes.

The method of any one of claims 1-3, wherein step (c) comprises incubating the redox reaction at 70-90°C for at least 30 seconds .

The method of any one of claims 1-4, wherein step (c) comprises incubating the redox reaction at 70-90°C for at least 1 minute.

The method of any one of claims 1-5, wherein step (c) comprises incubating the redox reaction at 80°C for at least 1 minute.

7. The method of any one of claims 1-6, wherein step (d) comprises stirring the redox reactant for at least 30 seconds.

The method of any one of claims 1-7, wherein step (d) comprises stirring the redox reactant for at least 1 minute.

The method of any one of claims 1-8 , wherein the agitation in step (d) comprises vortexing or sonication.

10. The method of any one of claims 1-9, wherein steps (a) to (d) are performed in less than 15 minutes.

11. The method of any one of claims 1-10, wherein steps (a) to (d) are performed in less than 5 minutes.

The method of any one of claims 1-11, wherein the biological sample comprises silica beads.

The method of any one of claims 1-12 , wherein the percarbonate comprises sodium percarbonate.

14. The method of any one of claims 1-13 , wherein said nuclease inhibitor comprises proteinase K.

15. The method of any one of claims 1-14 , wherein the chelating agent is EDTA .

16. The method of any one of claims 1-15, wherein said nucleic acid comprises DNA.

17. The method of any one of claims 1-16, wherein said nucleic acid comprises RNA.

18. The method of any one of claims 1-17, wherein the nucleic acid comprises a combination of DNA and RNA.

19. The method of any one of claims 1-18, wherein the nucleic acid is double-stranded.

20. The method of any one of claims 1-19, wherein the nucleic acid is single-stranded.

A method according to any one of claims 1 to 20, wherein said biological sample comprises bacterial cells, viruses, parasite cells and/or eukaryotic cells.

22. The method of claim 21, wherein said eukaryotic cells are plant cells, fungal cells, or mammalian cells.

23. The method of claim 22, wherein said fungal cells are yeast cells, mold cells, or mushroom cells.

23. The method of claim 22, wherein said mammalian cells are human, primate, or canine cells.

A method according to any one of claims 1 to 24, wherein said amplifiable nucleic acid is suitable for use as a template for PCR and/or RT-PCR.

26. The method of any one of claims 1-25 , further comprising (e) amplifying at least a portion of said amplifiable nucleic acid.

27. The method of claim 26 , wherein step (e) comprises amplifying at least a portion of said amplifiable nucleic acid by PCR or RT-PCR.