Sample preparation method and system
Method of preparing a biological sample appropriate for use in a subsequent in vitro nucleic acid amplification reaction. A biological sample is combined with an alkaline composition that lyses cells and denatures DNA to create a first liquid composition. The first liquid composition is then mixed with a buffer, a detergent, and a solid support that captures DNA to create a second liquid composition. The buffer, detergent, and solid support can be delivered as components of a single reagent. Captured DNA strands can be used as templates in subsequently performed nucleic acid amplification and detection reactions with improved sensitivity.
1. A method of processing a biological sample, the method comprising the steps of:
(a) mixing the biological sample with an alkaline composition that lyses cells and denatures DNA to create a first liquid composition,
wherein the first liquid composition has a pH in the range of from pH 12.0 to pH 13.5, and
wherein the first liquid composition is free of detergents;
(b) mixing the first liquid composition with a pH buffered detergent reagent to create a second liquid composition having a pH lower than pH 9.5,
wherein the pH buffered detergent reagent comprises a pH buffer, a detergent, and a solid support particle that captures DNA; and
(c) isolating the solid support particle, and any DNA captured thereon, from the second liquid composition.
2. The method of claim 1 , wherein the biological sample comprises viable bacterial cells, and wherein the method further comprises the step of culturing the viable bacterial cells to increase the number of bacterial cells.
3. The method of claim 1 , wherein the second liquid composition has a pH in the range of from pH 8.0 to pH 9.2.
4. The method of claim 1 , wherein each of steps (a)-(c) is conducted onboard an automated instrument that comprises a robotic fluid transfer device.
5. The method of claim 4 , further comprising the step of (d) performing an in vitro nucleic acid amplification reaction using DNA isolated in step (c) as templates, and detecting products of the in vitro nucleic acid amplification reaction.
6. The method of claim 5 , wherein detecting products of the in vitro nucleic acid amplification reaction indicates the presence of a species of Gram-positive bacteria in the biological sample.
7. The method of claim 5 , wherein step (d) is carried out onboard the automated instrument.
8. The method of claim 4 , wherein step (a) comprises combining the biological sample and the alkaline composition in a reaction vessel to create the first liquid composition, wherein step (b) comprises adding the pH buffered detergent reagent to the reaction vessel containing the first liquid composition to create the second liquid composition, and wherein each of steps (a) and (b) is performed using the robotic fluid transfer device of the automated instrument.
9. The method of claim 8 , wherein the automated instrument further comprises a transport mechanism that moves the reaction vessel from one position within the automated instrument to a different position within the automated instrument.
10. The method of claim 9 , wherein after step (b) and before step (c) there is the step of incubating the first liquid composition for a period of time between 1 minute and 10 minutes.
11. The method of claim 10 , wherein the incubating step comprises heating the first liquid composition.
12. The method of claim 10 , wherein the incubating step comprises transporting the reaction vessel from a first position in the automated instrument to a second position in the automated instrument, and wherein the second position in the automated instrument is at a temperature higher than the temperature at the first position in the automated instrument.
13. The method of claim 1 , wherein the solid support particle comprises a magnetically attractable particle.
14. The method of claim 1 , wherein the solid support particle in step (b) is a solid support particle that captures DNA independent of base sequence, and wherein step (c) comprises washing the solid support particle to remove any material not immobilized thereon, and then retaining the solid support particle after washing, whereby captured DNA is isolated.
15. The method of claim 1 , wherein the alkaline composition comprises a strong base in aqueous solution at a concentration of from 0.1 N to 2.2 N.
16. The method of claim 15 , wherein the pH of the first liquid composition is in the range of from pH 12.5 to pH 13.2.
17. The method of claim 15 , wherein the pH of the second liquid composition is in the range of from pH 7.6 to pH 8.8.
18. The method of claim 15 , wherein the alkaline composition comprises the strong base at a concentration of from 1.0 N to 1.7 N.
19. The method of claim 15 , wherein the strong base is selected from the group consisting of NaOH, KOH, and LiOH.
20. The method of claim 1 , wherein all steps are carried out under automated process control.