Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith
View Patent ↗An object of the present invention is to provide a novel primer and prove for detecting tubercle bacillus capable of avoiding false positive, and an easy-to-use, rapid and high-sensitivity method for detecting human tubercle bacillus ( Mycobacterium tuberculosis ) using the same. The present invention relates to an oligonucleotide comprising a part of or an entire sequence of the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or a part of or an entire sequence of the complementary sequence thereof, wherein the oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis , a primer and a probe containing the oligonucleotide for detecting Mycobacterium tuberculosis , and a method for detecting Mycobacterium tuberculosis using the primer and the probe.
1. An isolated or purified oligonucleotide consisting of a nucleic acid sequence selected from the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or the complementary sequence thereof, wherein the oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.
2. A primer for detecting Mycobacterium tuberculosis comprising a nucleic acid sequence selected from the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or the complementary sequence thereof, wherein the primer has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.
3. The primer according to claim 2 , wherein the primer is labeled with a labeling substance.
4. The primer according to claim 3 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and a biotin.
5. A probe for detecting Mycobacterium tuberculosis comprising a nucleic acid sequence selected from the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:11, or the complementary sequence thereof, wherein the probe has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.
6. The probe according to claim 5 , wherein the probe is labeled with a labeling substance.
7. The probe according to claim 6 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and a biotin.
8. The probe according to claim 6 , wherein the probe is labeled with a reporter fluorescent dye and with a quencher dye.
9. A kit for detecting Mycobacterium tuberculosis comprising:
at least one of a primer pair or a probe;
wherein the primer pair comprises a first primer comprising a nucleic acid sequence selected from the nucleic acid sequence described in SEQ ID NO:1 or SEQ ID NO:2, or the complementary sequence thereof, and a second primer comprising a nucleic sequence selected from the nucleic acid sequence described in SEQ ID NO:3 or SEQ ID NO:4, or the complementary sequence thereof;
wherein the probe comprises a nucleic acid sequence selected from the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:11, or the complementary sequence thereof; and
wherein the primer and the probe have a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.
10. The kit according to claim 9 , wherein the first or second primer is labeled with a labeling substance.
11. The kit according to claim 9 , wherein the probe is labeled with a labeling substance.
12. The kit according to claim 9 , comprising the primer pair and the probe, wherein the probe is labeled with a reporter fluorescent dye and with a quencher dye, and wherein the probe does not have the same nucleic acid sequence as the first and second primers.
13. The probe according to claim 8 , wherein the 5′-terminal is labeled with a reporter fluorescent dye and the 3′-terminal is labeled with a quencher dye.
14. The oligonucleotide according to claim 1 , wherein the oligonucleotide is obtained using a DNA synthesizer.
15. The primer according to claim 2 , wherein the primer comprises an oligonucleotide portion consisting of the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or the complementary sequence thereof.
16. The primer according to claim 15 , further comprising a labeling substance.
17. The probe according to claim 5 , wherein the probe comprises an oligonucleotide portion consisting of the nucleic acid sequence described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:11, or the complementary sequence thereof.
18. The probe according to claim 17 , further comprising a labeling substance.
19. The probe according to claim 18 , wherein the probe is labeled with a labeling substance selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and a biotin.
20. The probe according to claim 17 , wherein the probe is labeled with a reporter fluorescent dye and with a quencher dye.
21. The probe according to claim 20 , wherein the 5′-terminal is labeled with a reporter fluorescent dye and the 3′-terminal is labeled with a quencher dye.
22. The kit according to claim 12 , wherein the primer pair comprises the first primer comprising an oligonucleotide portion consisting of the nucleic acid sequence described in SEQ ID NO:1 and the second primer comprising an oligonucleotide portion consisting of the nucleic sequence described in SEQ ID NO:3, and wherein the probe comprises an oligonucleotide portion consisting of the nucleic acid sequence described in SEQ ID NO:11.