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

• US 8,628,926 B2
• Filed: 10/25/2011
• Issued: 01/14/2014
• Est. Priority Date: 04/26/2004
• Status: Expired due to Fees

• 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
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/281,421
• Publication Number:
  US 20130109013A1
• Time in Patent Office: 812 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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

Primers and probes are provided for selectively detecting tubercle bacillus, and methods for detecting low copy numbers of human tubercle bacillus (Mycobacterium tuberculosis) using the same are presented. Oligonucleotides 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:S, 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 are disclosed, wherein the oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis. Primers and probes containing such oligonucleotides, and methods for detecting Mycobacterium tuberculosis using the primers and the probes are presented.

Claims

1. A method for selectively detecting Mycobacterium tuberculosis and not detecting other Mycobacteria species, comprising using a primer comprising an oligonucleotide comprising a nucleotide sequence selected from 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 oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.

2. The method according to claim 1, further comprising using a probe comprising an oligonucleotide comprising a nucleotide sequence selected from 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 oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.

3. The method according to claim 1, comprising:

performing a nucleic acid amplification reaction with the primer and with a nucleic acid in a sample as a template; and

detecting an obtained primer extension product.

4. The method according to claim 3, further comprising:

performing electrophoresis of the primer extension product obtained in the nucleic acid amplification reaction; and

determining the presence of Mycobacterium tuberculosis in the sample on the basis of the obtained electrophoresis result.

5. The method according to claim 4, wherein the nucleic acid amplification reaction is performed using:

(1) a first primer comprising the oligonucleotide selected from a nucleic acid sequence consisting of SEQ ID NO;

1 or SEQ ID NO;

2, or the complementary sequence thereof; and

(2) a second primer comprising the oligonucleotide selected from a nucleic acid sequence consisting of SEQ ID NO;

3 or SEQ ID NO;

4, or the complementary sequence thereof.

6. The method according to claim 4, wherein the presence of Mycobacterium tuberculosis is determined based on the primer extension product having an expected number of base pairs.

7. The method according to claim 6, wherein the nucleic acid amplification reaction is performed using a first primer comprising the oligonucleotide consisting of SEQ ID NO:

1 and a second primer comprising the oligonucleotide consisting of SEQ ID NO;

3; and

the primer extension product of 178 base pairs determines the presence of Mycobacterium tuberculosis in the sample.

8. The method according to claim 6, wherein the nucleic acid amplification reaction is performed using a first primer comprising the oligonucleotide consisting of SEQ ID NO:

2 and a second primer comprising the oligonucleotide consisting of SEQ ID NO;

4; and

the primer extension product having 182 base pairs determines the presence of Mycobacterium tuberculosis in the sample.

9. The method according to claim 6, wherein the nucleic acid amplification reaction is performed using a first primer comprising the oligonucleotide consisting of SEQ ID NO:

1 and a second primer comprising the oligonucleotide consisting of SEQ ID NO;

4; and

the primer extension product having 193 base pairs determines the presence of Mycobacterium tuberculosis in the sample.

10. The method according to claim 3, further comprising:

performing electrophoresis of the primer extension product obtained in the nucleic acid amplification reaction;

hybridizing with the obtained primer extension product a probe comprising an oligonucleotide selected from a nucleotide sequence consisting of 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 a label selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or biotin, wherein the oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis; and

detecting a signal derived from the hybridized labeled probe, wherein the presence of Mycobacterium tuberculosis is determined based on the hybridization of the labeled probe to the obtained primer extension product.

11. The method according to claim 3, further comprising:

performing electrophoresis of the primer extension product obtained in the nucleic acid amplification reaction;

hybridizing with the obtained primer extension product a probe comprising an oligonucleotide 20 or more bases in length designed from a nucleotide sequence selected from SEQ ID NO;

6, SEQ ID NO;

7, or SEQ ID No;

8, or the complementary sequence thereof, and a label, wherein the oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis; and

detecting a signal derived from the hybridized labeled probe, wherein the presence of Mycobacterium tuberculosis is determined based on the hybridization of the labeled probe to the obtained primer extension product.

12. The method according to claim 11, wherein the label is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or biotin.

13. The method according to claim 11, wherein the nucleic acid amplification reaction is performed using a first primer comprising the oligonucleotide consisting of SEQ ID NO:

1 and a second primer comprising the oligonucleotide consisting of SEQ ID NO;

3; and

the labeled probe comprises the oligonucleotide 20 or more bases in length designed from SEQ ID NO;

6 or the complementary sequence thereof.

14. The method according to claim 11, wherein the nucleic acid amplification reaction is performed using a first primer comprising the oligonucleotide consisting of SEQ ID NO:

2 and a second primer comprising the oligonucleotide consisting of SEQ ID NO;

4; and

the labeled probe comprises the oligonucleotide 20 or more bases in length designed from SEQ ID NO;

7 or the complementary sequence thereof.

15. The method according to claim 3, further comprising:

using a probe comprising an oligonucleotide 20 or more bases in length designed from a nucleotide sequence selected from 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, wherein the probe is labeled with a 5′

-terminal reporter fluorescent dye and a 3′

-terminal quencher dye; and

detecting 5′

-terminal reporter fluorescent dye released from the labeled probe.

16. The method according to claim 3, wherein the primer comprises a label, the method further comprising detecting the label of the labeled primer incorporated into the obtained primer extension product.

17. The method according to claim 16, further comprising, after performing the nucleic acid amplification reaction, separating free labeled primer from the primer extension product.

18. The method according to claim 17, wherein separating the free labeled primer comprises precipitating the primer extension product and removing the supernatant liquid containing the free labeled primer from the precipitated primer extension product.

19. The method according to claim 17, wherein separating the free labeled primer comprises performing gel chromatography.

20. A method for selectively detecting Mycobacterium tuberculosis and not detecting other Mycobacteria species, comprising using a probe comprising an oligonucleotide portion consisting of the nucleotide sequence selected from 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 oligonucleotide has a property of hybridizing with the nucleic acid sequence of IS6110 gene in Mycobacterium tuberculosis.