Nucleic acid detection method
The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.
1. A method for detecting the presence of a single stranded target nucleic acid of defined sequence in a sample comprising:
a) contacting the sample with:
(i) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5′ to 3′ direction one strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridising to a first hybridisation sequence in the target nucleic acid, and said second primer comprises in the 5′ to 3′ direction one strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridising to the reverse complement of a second hybridisation sequence upstream of the first hybridisation sequence in the target nucleic acid;
(ii) a strand displacement DNA polymerase;
(iii) dNTPs;
(iv) one or more modified dNTP;
(v) a first restriction enzyme that is not a nicking enzyme but is capable of recognising the recognition sequence of the first primer and cleaving only the first primer strand of the cleavage site when said recognition sequence and cleavage site are double stranded, the cleavage of the reverse complementary strand being blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTP; and
(vi) a second restriction enzyme that is not a nicking enzyme but is capable of recognising the recognition sequence of the second primer and cleaving only the second primer strand of the cleavage site when said recognition sequence and cleavage site are double stranded, the cleavage of the reverse complementary strand being blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTP;
to produce, without temperature cycling, in the presence of said target nucleic acid, amplification product;
b) contacting the amplification product of step a) with:
(i) a first oligonucleotide probe which is capable of hybridising to a first single stranded detection sequence in at least one species within the amplification product and which is attached to a moiety that permits its detection; and
(ii) a second oligonucleotide probe which is capable of hybridising to a second single stranded detection sequence upstream or downstream of the first single stranded detection sequence in said at least one species within the amplification product and comprises a repeat motif sequence comprising 3 or more repeat copies of a 2 to 4 base DNA sequence motif;
wherein one of the first and second oligonucleotide probes is blocked at the 3′ end from extension by the DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, and wherein the blocked oligonucleotide probe is contacted with the sample simultaneously to the performance of step a);
and where hybridisation of the first and second probes to said at least one species within the amplification product produces a detector species; and
c) detecting the presence of the detector species produced in step b) by contacting the detector species with a substrate having an immobilised oligonucleotide capture probe comprising a single stranded hybridisation sequence complementary to the repeat motif sequence, wherein the presence of the detector species indicates the presence of the target nucleic acid in said sample.
2. The method according to claim 1 , wherein the blocked oligonucleotide probe is rendered not capable of being cleaved by either the first or second restriction enzymes due to the presence of one or more sequence mismatch(es) and/or one or more modification(s).
3. The method according to claim 2 , wherein the one or more modification(s) are phosphorothioate linkage(s).
4. The method according to claim 1 , wherein the blocked oligonucleotide probe comprises an additional region such that the 3′ end of the species within the amplification product to which the blocked oligonucleotide probe hybridises can be extended by the strand displacement DNA polymerase.
5. The method according to claim 1 , wherein the one or more modified dNTP is an alpha thiol modified dNTP.
6. The method according to claim 5 , wherein the first and second hybridisation sequences in the target nucleic acid are separated by 3 to 15 bases and wherein in step b) either the first or second single stranded detection sequence in the at least one species within the amplification product includes at least 3 bases of the sequence corresponding to the 3 to 15 bases separating the first and second hybridisation sequences in the target nucleic acid.
7. The method according to claim 1 , wherein the first and second restriction enzyme are the same restriction enzyme.
8. The method according to claim 1 , wherein the moiety that permits the detection of the first oligonucleotide probe, is a colorimetric or fluorometric dye or a moiety that is capable of attachment to a colorimetric or fluorometric dye.
9. The method according to claim 8 , wherein the moiety that is capable of attachment to a colorimetric or fluorometric dye is biotin.
10. The method according to claim 1 , wherein the detector species is detected by a change in electrical signal.
11. The method according to claim 1 , wherein the presence of the detector species is detected by nucleic acid lateral flow, wherein the nucleic acid lateral flow comprises the oligonucleotide capture probe immobilized on the lateral flow strip.
12. The method according to claim 1 , wherein step c) produces a colorimetric or electrochemical signal using carbon or gold.
13. The method according to claim 1 , wherein the first and second hybridisation sequences in the target nucleic acid are separated by 0 to 15 bases.
14. The method according to claim 1 , wherein the target nucleic acid is selected from
(i) single stranded RNA, including single stranded RNA derived from double stranded RNA and single stranded RNA derived from double stranded DNA; or
(ii) single stranded DNA, including single stranded DNA derived from single stranded RNA including by use of a reverse transcriptase, single stranded DNA derived from double stranded DNA by strand invasion, and single stranded DNA derived from double stranded DNA by use of a nuclease.
15. The method according to claim 1 , wherein the presence of two or more different target nucleic acids of defined sequence are detected in the same sample.
16. The method according to claim 1 , wherein the sample is a biological sample, including a nasal or nasopharyngeal swab or aspirate, blood or a sample derived from blood, urine, a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample or a biodefence sample.
17. The method according to claim 1 , wherein the target nucleic acid is viral or derived from viral nucleic acid material, is bacterial or derived from bacterial nucleic acid material, is circulating, cell-free DNA released from cancer cells or foetal cells, is micro RNA or derived from micro RNA.
18. The method according to claim 1 , wherein the target nucleic acid is derived from a pathogen.
19. The method of claim 18 , wherein the pathogen is viral.
20. The method of claim 19 , wherein the pathogen is HIV, influenza, RSV, Rhinovirus, norovirus, HPV, Ebola, or Epstein-Barr virus.
21. The method of claim 18 , wherein the pathogen is bacterial.
22. The method of claim 21 , wherein the pathogen is MRSA or Clostridium difficile.
23. The method of claim 18 , wherein the pathogen is the relevant causative pathogen of an infectious disease.
24. The method of claim 23 , wherein the infectious disease is meningitis, hepatitis, malaria, tuberculosis, plague, polio, chlamydia, herpes, gonorrhoea, measles, mumps, rubella, cholera or smallpox.
25. The method according to claim 1 , wherein both the first and second oligonucleotide probes are blocked at the 3′ from extension by the DNA polymerase and are not capable of being cleaved by either the first or second restriction enzymes.
26. The method of claim 1 , wherein the target nucleic acid is derived from a cancer.
27. The method of claim 26 , wherein the cancer is colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukaemia, esophageal cancer, ovarian cancer, kidney cancer, stomach cancer or melanoma.