Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith

• US 8,044,184 B2
• Filed: 04/22/2005
• Issued: 10/25/2011
• Est. Priority Date: 04/26/2004
• Status: Active Grant

• Current Assignee: FUJIFILM Wako Pure Chemical Corporation (Fujifilm Holdings Corporation)
• Original Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Inventors: Ishikawa, Tomokazu
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US11/587,510
• Publication Number:
  US 20080241826A1
• Time in Patent Office: 2,377 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y10S 435/975 Kit

Abstract

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.

Claims

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.

14. The oligonucleotide according to claim 1, wherein the oligonucleotide is obtained using a DNA synthesizer.

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.

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.

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.

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.

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.

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.

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.