Rapid extraction of nucleic acids from clinical samples for downstream applications
View Patent ↗Disclosed herein are novel methods and compositions for rapidly extracting and amplifying nucleic acids from a sample where the sample is combined with an extraction reagent comprising a reducing agent to form a mixture and incubating said mixture at ambient temperature for a period of time not exceeding 30 minutes to generate a nucleic acid extract. In certain embodiments of the method, the nucleic acid extract is subjected to a nucleic acid amplification reaction. In certain aspects, oligonucleotide primers specific for nucleic acids of Chlamydia species and/or Neisseria species are added prior to initiating the amplification reaction.
1. A method for preparing a nucleic acid extract from a sample comprising cells, the method comprising:
obtaining the sample;
combining the sample with an extraction reagent, said reagent comprising a reducing agent, and optionally a buffer, to form a mixture;
sealing said mixture within a reaction vessel;
incubating said mixture in said sealed reaction vessel at ambient temperature for a period of time not exceeding 30 minutes to generate a nucleic acid extract; and
combining said mixture with a nucleic acid amplification reagent under conditions that promote nucleic acid amplification of said nucleic acid extract.
2. The method of claim 1 , wherein said reducing agent is selected from the group consisting of dithiothreitol (DTT) and beta mercapto-ethanol (β-ME).
3. The method of claim 2 , wherein said reducing agent is present at a concentration less than or equal to 40 mM.
4. The method of claim 1 , wherein said extraction reagent comprises a buffer, and wherein said buffer provides a buffering capacity within said mixture that is less than a buffering capacity of 50 mM Tris at pH8.5.
5. The method of claim 4 , wherein said buffer comprises DTT and wherein the concentration of said DTT is 1 mM to 40 mM.
6. The method of claim 1 , wherein said ambient temperature ranges from 15° C. to 32° C.
7. The method of claim 1 , wherein said period of time does not exceed 10 minutes.
8. The method of claim 7 , wherein said period of time does not exceed 5 minutes.
9. The method of claim 1 , wherein said sample is suspected to comprise bacteria.
10. The method of claim 9 , wherein said bacteria comprise cysteine-rich cell walls.
11. The method of claim 9 , wherein said bacteria are selected from the group consisting of all Chlamydia species and strains and all Neisseria species and strains.
12. The method of claim 1 , wherein said amplification is initiated not later than 10 minutes following the conclusion of said incubation step.
13. The method of claim 1 , wherein said nucleic acid amplification occurs via a reaction selected from the group consisting of a polymerase chain reaction (PCR), a loop-mediated isothermal amplification (LAMP), a strand displacement amplification, a multiple displacement amplification, a recombinase polymerase amplification, a helicase dependent amplification and a rolling circle amplification.
14. The method of claim 1 , wherein said nucleic acid amplification occurs via an isothermal reaction.
15. The method of claim 14 , wherein said nucleic acid amplification is a loop-mediated isothermal amplification reaction.
16. The method of claim 1 , wherein said extraction reagent comprises a detergent.
17. The method of claim 1 , wherein said sample is selected from the group consisting of an endocervical swab, vaginal swab, urethral swab, pharyngeal swab, conjunctival swab and rectal swab or from remnant transport media from any of these types of swab samples.