Enhanced methods of ribonucleic acid hybridization

Patent metadata

Field	Value
Publication number	US-10253353-B2
Application number	US-201415101676-A
Country	US
Kind code	B2
Filing date	Dec 5, 2014
Priority date	Dec 6, 2013
Publication date	Apr 9, 2019
Grant date	Apr 9, 2019

Abstract

This disclosure relates to a method for increasing the hybridization efficiency of a probe and a target RNA in a sample, for example to identify a particular RNA present in the sample. The method includes heating a lysate sample comprising at least one target RNA, such as a tRNA, mRNA or rRNA, at a temperature of about 95° C. for a time sufficient to interfere with secondary structure of the RNA, wherein the time is short enough, such that the RNA in the cell lysate sample are not significantly degraded, and wherein the lysate comprises a cell lysis buffer comprising a chemical denaturant. To detect a target RNA in the lysate, the lysate is contacted with at least one detectable probe, such as a labeled probe, designed to specifically hybridize to the target RNA in the lysate.

First claim

We claim:

1. A method of increasing the hybridization efficiency of a probe and a target RNA in a cell lysate, comprising:
heating a cell lysate sample comprising at least one target RNA at a temperature of between 80° C. and 95° C. for a time sufficient to interfere with secondary structure of the RNA, wherein the time is short enough, such that the RNA in the cell lysate sample is not sufficiently degraded to prevent hybridization, and wherein the cell lysate comprises a cell lysis buffer comprising a chemical denaturant;
contacting the cell lysate sample with at least one detectable probe that specifically hybridizes to the at least one target RNA in the cell lysate sample; and
detecting hybridization between the probe and the at least one target RNA, wherein the detected hybridization between the probe and the at least one target RNA is increased at least 2.5 fold relative to the hybridization between the probe and the at least one target RNA in the absence of the heating step; and
wherein the at least one target RNA is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa , and Staphylococcus aureus.

2. The method of claim 1 , wherein the lysis buffer comprises a guanidine salt.

3. The method of claim 2 , wherein the guanidine salt comprises guanidine isothiocyanate.

4. The method of claim 1 , wherein the lysis buffer comprises an RNAse inactivator.

5. The method of claim 4 , wherein the RNAse inactivator comprises a mercaptan, or metal chelation agent.

6. The method of claim 5 , wherein the mercaptan comprises B-mercaptoethanol.

7. The method of claim 1 , further comprising, centrifugation of the sample prior to contacting the cell lysate sample with the probe, to remove denatured protein and other cellular debris.

8. The method of claim 1 , wherein the temperature of the sample is maintained at at least 65° C. prior to contact with the probe.

9. The method of claim 1 , wherein the at least one target RNA comprises target ribosomal RNA (rRNA), target messenger RNA (mRNA), or a combination thereof.

10. The method of claim 1 , wherein the detectable probe is labeled.

11. The method of claim 10 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled.

12. The method of claim 10 , wherein the label comprises a capture moiety.

13. The method of claim 12 , wherein the capture moiety comprises biotin linked to a nucleotide.

14. The method of claim 1 , wherein the probe is attached to a solid surface.

15. The method of claim 1 , further comprising:
distinguishing between two or more species of one or more organisms in a sample, comprising:
contacting the sample with a set of probes, wherein the set of probes contains at least one detectable probe that is specific for a target RNA sequence of each species to be tested, and wherein individual probes in the probe set are specific for each species; and
detecting hybridization between one or more of the probes and the RNA, thereby distinguishing between two or more species in the sample.

16. The method of claim 15 , further comprising contacting the sample with a second set of probes, wherein the second set of probes contains at least one second detectable probe that is specific for the target RNA and wherein individual probes in the second set of probes bind to substantially the same region of the RNA as the first set of probes, but wherein the second set of probes does not align in sequence with the first set of probes.

17. The method of claim 16 , wherein the second detectable probe is labeled.

18. The method of claim 17 , wherein the second detectable probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled.

19. The method of claim 16 , wherein the second detectable probe is attached to a solid surface.

20. The method of claim 17 , wherein the label comprises a capture moiety.

21. The method of claim 20 , wherein the capture moiety comprises biotin linked to a nucleotide.

22. The method of claim 1 , wherein the sample is obtained from an animal subject suspected of having an infection with one or more organisms pathogenic to that animal subject.

23. The method of claim 22 , wherein the animal subject is a veterinary subject.

24. The method of claim 22 , wherein the animal subject is a human subject.

25. The method of claim 1 , wherein detecting hybridization between the probe and the RNA indicates the presence of at least one of the two or more species in the sample.

26. The method of claim 25 , wherein the presence of at least one of the two or more species indicates the sample is infected or contaminated with the at least one species of organism.

27. The method of claim 1 , wherein the sample is obtained from an environmental sample.

28. The method of claim 1 , wherein the sample is obtained from a food product or material that will become a food product.

29. The method of claim 28 , wherein the material that will become a food product comprises meat, poultry, or plant matter.

Assignees

• Broad Inst Inc
• Massachusetts Gen Hospital

Inventors

• Bhattacharyya Roby
• Hung Deborah
• Patel Milesh

Classifications

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q2523/113

  Denaturating agents · CPC title

• C12Q2527/101

  Temperature · CPC title

• C12Q2527/113

  Time · CPC title