Method for obtaining and initiating amplification of a target nucleic acid sequence
View Patent ↗The present invention relates to oligonucleotides useful for determining the presence of Mycobacterium tuberculosis complex organisms in a test sample. The oligonucleotides of the present invention may be incorporated into detection probes, helper probes, capture probes and amplification oligonucleotides, and used in various combinations thereof.
1. A method for obtaining and initiating amplification of a target nucleic acid sequence from a mycobacterial organism present in a sample, the method comprising the steps of:
a) exposing a sample to a lytic composition for about 15 minutes to at least, or about, 60 minutes under conditions sufficient to kill and lyse said organism that include a temperature of about 60° C. to at least, or about, 95° C., thereby releasing a target nucleic acid into a lysate, the lytic composition comprising a detergent, an amplification oligonucleotide for amplifying a target nucleic acid sequence contained in the target nucleic acid, a solid support comprising a plurality of magnetically charged particles or beads, and a capture probe for immobilizing the target nucleic acid on the solid support;
b) forming a hybrid complex in the lysate, which hybrid complex comprises the capture probe, the target nucleic acid and the amplification oligonucleotide;
c) immobilizing the hybrid complex on the solid support and removing components of the lysate which are not part of the hybrid complex formed in step b); and
d) exposing the target nucleic acid to amplification conditions, such that the amplification oligonucleotide is enzymatically extended to form a complementary copy of the target nucleic acid sequence.
2. The method of claim 1 , wherein the temperature of step b) is lower than the temperature of step a).
3. The method of claim 1 , wherein the sample is exposed to the lytic composition for at least, or about, 15 minutes under conditions that include a temperature of at least, or about, 95° C.
4. The method of claim 1 , wherein the amplification oligonucleotide comprises a promoter sequence which is recognized by an RNA polymerase.
5. The method of claim 1 , wherein the detergent is a cationic detergent.
6. The method of claim 5 , wherein the detergent is lithium lauryl sulfate.
7. The method of claim 5 , wherein the detergent is present at a concentration of from about 0.1 to about 5% (v/v) in step a).
8. The method of claim 5 , wherein the detergent is present at a concentration of from about 0.1 to about 3% (v/v) in step a).
9. The method of claim 5 , wherein the detergent is present at a concentration of from about 0.1 to about 1.5% (v/v) in step a).
10. The method of claim 5 , wherein the detergent is present at a concentration of from about 0.1 to about 0.3% (v/v) in step a).
11. The method of claim 1 , wherein mechanical means are not used to lyse the organism.
12. The method of claim 1 , wherein sonication is not used to lyse the organism.
13. The method of claim 1 , wherein the salt concentration of steps a)-c) is from about 0.6 M to about 0.9 M.
14. The method of claim 1 , wherein the sample is a respiratory sample.
15. The method of claim 14 , wherein the respiratory sample is sputum.
16. The method of claim 1 , where the mycobacterial organism is a Mycobacterium tuberculosis complex organism.