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 comprising:
incubating a mixture comprising (i) a sample suspected of comprising bacteria and (ii) an extraction reagent at ambient temperature for a period of time not exceeding 30 minutes to generate a nucleic acid extract,
wherein the reagent comprises a reducing agent, and optionally a buffer; and
combining the mixture comprising the nucleic acid extract with a nucleic acid amplification reagent under conditions that promote nucleic acid amplification of the nucleic acid extract.
2. The method of claim 1 , wherein the reducing agent is dithiothreitol (DTT).
3. The method of claim 2 , wherein the DTT is present at a concentration of 1 mM to 40 mM.
4. The method of claim 1 , wherein the reducing agent is beta mercapto-ethanol (β-ME).
5. The method of claim 4 , wherein the β-ME is present at a concentration less than or equal to 40 mM.
6. The method of claim 1 , wherein the extraction reagent comprises a buffer, and wherein the buffer provides a buffering capacity within the mixture that is less than a buffering capacity of 50 mM Tris at pH8.5.
7. The method of claim 1 , wherein the extraction reagent comprises a buffer comprising Tris at a concentration of 1.6 mM.
8. The method of claim 1 , wherein the ambient temperature ranges from 15° C. to 32° C. and the period of time does not exceed 20 minutes or 10 minutes.
9. The method of claim 1 , wherein the ambient temperature ranges from 15° C. to 32° C. and the period of time does not exceed 2 minutes or 5 minutes.
10. The method of claim 1 , wherein the bacteria comprise cysteine-rich cell walls.
11. The method of claim 1 , wherein the 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 the nucleic acid amplification is initiated not later than 10 minutes following the conclusion of the incubation step.
13. The method of claim 1 , wherein the nucleic acid amplification is initiated not later than 5 minutes or no later than 2 minutes following the conclusion of the incubation step.
14. The method of claim 1 , wherein the 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.
15. The method of claim 1 , wherein the nucleic acid amplification occurs via an isothermal reaction.
16. The method of claim 1 , wherein the extraction reagent comprises a detergent.
17. The method of claim 1 , wherein the extraction reagent comprises a PCR enhancer, wherein the PCR enhancer is betaine or trimethylglycine.
18. The method of claim 1 , wherein the extraction reagent comprises a magnesium salt.
19. The method of claim 1 , wherein the 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.