Alkaline shock-based method of processing a biological sample
View Patent ↗Method of processing a biological sample to yield nucleic acid appropriate for use in a subsequent in vitro nucleic acid amplification reaction. The method involves a rapid, transient exposure to alkaline conditions which can be achieved by mixing an alkaline solution with a pH-buffered solution that includes a detergent and the biological sample to be tested for the presence of particular nucleic acid species using in vitro amplification. The invented method advantageously can improve detection of some target nucleic acids without substantially compromising detectability of others. The method is particularly useful for simultaneously preparing RNA and DNA templates that can be used in multiplex amplification reactions.
1. A method of processing a biological sample, comprising the steps of:
(a) combining said biological sample with a pH buffer and a detergent that lyses or disrupts biological membranes, whereby there is created a first liquid composition having a first pH;
(b) mixing with said first liquid composition an alkaline composition, whereby there is created a second liquid composition having a second pH,
wherein said second pH is at least 0.2 pH units higher than said first pH,
wherein said second pH is lower than pH 9.5,
wherein said alkaline composition comprises a strong base in solution at a concentration of at least 0.1 N, and
wherein both DNA and RNA that may be present in said biological sample are substantially stable and not degraded or hydrolyzed in said second liquid composition; and
(c) purifying nucleic acids, comprising RNA, from said second liquid composition.
2. The method of claim 1 , wherein step (c) comprises purifying nucleic acids, comprising DNA, from said second liquid composition.
3. The method of claim 1 , wherein said pH buffer and said detergent in step (a) are each components of a buffered detergent solution, and wherein step (a) comprises combining said biological sample with an aliquot of said buffered detergent solution.
4. The method of claim 1 , wherein said strong base is selected from the group consisting of NaOH, LiOH and KOH.
5. The method of claim 1 , wherein said concentration of at least 0.1 N in step (b) is a concentration in the range of from 0.1 N to 2.2 N.
6. The method of claim 1 , wherein purifying step (c) comprises,
(i) immobilizing nucleic acids from said second liquid composition onto a solid support by sequence-specific target capture;
(ii) washing the solid support to remove any material not immobilized thereon; and
(iii) retaining the solid support after washing, whereby nucleic acids immobilized thereon are purified.
7. The method of claim 6 , further comprising the step of:
(d) performing an in vitro nucleic acid amplification reaction using nucleic acids purified in step (c) as templates.
8. The method of claim 1 , wherein purifying step (c) comprises,
(i) immobilizing nucleic acids from said second liquid composition onto a solid support by non-specific target capture that is substantially independent of base sequence;
(ii) washing the solid support to remove any material not immobilized thereon; and
(iii) retaining the solid support after washing, whereby nucleic acids immobilized thereon are purified.
9. The method of claim 8 , further comprising the step of:
(d) performing an in vitro nucleic acid amplification reaction using nucleic acids purified in step (c) as templates.
10. The method of claim 1 , wherein purifying step (c) comprises,
(i) capturing onto a solid support nucleic acids from said second liquid composition;
(ii) washing the solid support to remove any material not immobilized thereon, and
(iii) retaining the solid support after washing, whereby nucleic acids captured thereon are purified.
11. The method of claim 10 , further comprising the step of:
(d) performing an in vitro nucleic acid amplification reaction using nucleic acids purified in step (c) as templates.
12. The method of claim 11 , wherein said in vitro nucleic acid amplification reaction comprises a reverse transcriptase.
13. The method of claim 11 , wherein said in vitro nucleic acid amplification reaction is a multiplex in vitro nucleic acid amplification reaction that amplifies an RNA target and a DNA target.
14. The method of claim 13 , wherein said RNA target is selected from the group consisting of an HIV-1 target nucleic acid and an HCV target nucleic acid.
15. The method of claim 13 , wherein said DNA target is an HBV target nucleic acid.
16. The method of claim 10 , wherein nucleic acids captured onto the solid support comprise RNA and DNA.
17. The method of claim 10 , wherein said second pH is in the range of from pH 8.0 to pH 9.2.
18. The method of claim 10 , wherein said second pH is pH 9.2 or lower.
19. The method of claim 10 , wherein said second pH is in the range of pH 8.0 to pH 8.7.
20. The method of claim 10 , wherein said first pH is in the range of from pH 6.5 to pH 8.0, and wherein said second pH is in the range of from pH 8.2 to pH 9.2.
21. The method of claim 1 , further comprising the step of:
(d) performing an in vitro nucleic acid amplification reaction using nucleic acids purified in step (c) as templates.
22. The method of claim 21 , wherein said in vitro nucleic acid amplification reaction comprises a reverse transcriptase.
23. The method of claim 21 , wherein said in vitro nucleic acid amplification reaction is a multiplex in vitro nucleic acid amplification reaction that amplifies an RNA target and a DNA target.
24. The method of claim 23 , wherein said RNA target is selected from the group consisting of an HIV-1 target nucleic acid and an HCV target nucleic acid.
25. The method of claim 23 , wherein said DNA target is an HBV target nucleic acid.