PRIMER AND PROBE FOR DETECTION OF MYCOBACTERIUM AVIUM AND METHOD FOR DETECTION OF MYCOBACTERIUM AVIUM BY USING THE PRIMER OR PROBE

1 - 21 . (canceled)

22 . A primer for detecting Mycobacterium avium consisting of an oligonucleotide comprising a nucleotide sequence selected from SEQ ID NO:175, 176, 177, 178 181, and 182,

wherein the primer is not more than about 50 nucleotides in length;

wherein the primer is labeled with a labeling substance, which is bound via a covalent bond or a linker; and

wherein the primer is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome.

23 . The primer according to claim 22 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin.

24 . The primer according to claim 22 , wherein the primer consists of a primer pair consisting of a first primer and a second primer;

wherein the first primer is not more than about 50 nucleotides in length and comprises an oligonucleotide that comprises the nucleotide sequence of SEQ ID NO: 175, 177, or 181;

wherein at least one of the first primer or the second primer is labeled with a labeling substance, which is bound via a covalent bond or a linker; and

wherein the first primer and the second primer are together capable of amplifying a genomic DNA fragment of Mycobacterium avium genome.

25 . The primer pair according to claim 24 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin.

26 . The primer pair according to claim 24 , wherein the second primer of said primer pair is not more than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 176, 178, or 182.

27 . A method for detecting Mycobacterium avium , characterized in that an oligonucleotide which comprises a part or an entire 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, or a part or an entire of the sequence complementary to 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, and which is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome is used as a primer and/or a probe.

28 . The method according to claim 27 , characterized in that the nucleic acid amplification reaction is carried out using as a primer an oligonucleotide which comprises a part or an entire 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, or a part or an entire of the sequence complementary to 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, and which is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome, and using nucleic acid in a sample as a template, and the obtained primer extension product is detected.

29 . The method according to claim 28 , wherein a labeled probe which is labeled with a labeling substance is further used.

30 . The method according to claim 27 , characterized by comprising the following steps:

(1) carrying out a nucleic acid amplification reaction using as a primer an oligonucleotide which comprises a part or an entire 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, or a part or an entire of the sequence complementary to 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, and which is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome, and using the nucleic acid in a sample as a template; and

(2) carrying out an electrophoresis of the primer extension product obtained in (1), and determining the presence of Mycobacterium avium based on the result of the electrophoresis.

31 . The method according to claim 30 , wherein the sample is determined as positive for Mycobacterium avium in either one of the following cases:

(1) a case when, after electrophoresis, the electrophoretic fraction obtained is examined for the presence of a primer extension product having objective base pair size, and the primer extension product having the objective number of base pair is confirmed;

(2) a case when, after electrophoresis, a hybridization of the electrophoretic fraction obtained is carried out with a labeled probe prepared by labeling an oligonucleotide which comprises a part or an entire 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, or a part or an entire of the sequence complementary to 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, and which is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome with a labeling substance, and a fraction hybridized with the labeled probe is confirmed by detecting a signal derived from the labeled probe.

32 . The method according to claim 27 , wherein the primer has been labeled with a labeling substance; and wherein the polymerase chain reaction is carried out using the primer and the nucleic acid in a sample as a template, thereafter a signal derived the obtained primer extension product is measured.

33 . The method according to claim 32 , wherein, after the nucleic acid amplification chain reaction is carried out, and free labeled primer is removed, then a signal derived the primer extension product is measured.

34 . The method according to claim 27 , characterized in that, an oligonucleotide which comprises a part or an entire 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, or a part or an entire of the sequence complementary to 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135 and SEQ ID NO:136, and which is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome, is labeled with a labeling substance and used as a labeled probe; the labeled probe is hybridized with the nucleic acid in a sample; free labeled probe is removed; and then a signal derived from the hybridized complex is detected.

35 . A reagent kit comprising:

(i) a primer for detecting Mycobacterium avium consisting of an oligonucleotide comprising a nucleotide sequence selected from SEQ ID NO:175, 176, 177, 178, 181, and 182:

wherein the primer is not more than about 50 nucleotides in length;

wherein the primer is optionally labeled with a labeling substance, which is bound via a covalent bond or a linker; and

wherein the primer is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome: and

(ii) (1) a nucleic acid synthetase and/or (2) a substrate for a nucleic acid synthetase and/or (3) a double strand intercalator and/or (4) a signal detection substance.

36 . The reagent kit according to claim 35 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin.

37 . The reagent kit according to claim 35 , further comprising:

a probe, wherein the probe comprises (a) an oligonucleotide that comprises a nucleotide sequence selected from:

SEQ ID NO: 136, or a full nucleotide sequence complementary thereto, wherein the probe is no longer than about 1100 nucleotides; or

SEQ ID NO: 202, 203, 205, or a full nucleotide sequence complementary thereto; or

20-50 consecutive nucleotides of SEQ ID NO: 202, 203, 205, or a full nucleotide sequence complementary thereto; and

optionally (b) a labeling substance, which is bound to the oligonucleotide via a covalent bond or a linker; and wherein the probe is capable of hybridizing with a genomic DNA fragment of Mycobacterium avium genome.

38 . The reagent kit according to claim 37 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin.

39 . The reagent kit according to claim 35 , wherein the primer is a primer pair consisting of a first primer and a second primer;

wherein the first primer is not more than about 50 nucleotides in length and comprises an oligonucleotide that comprises the nucleotide sequence of SEQ ID NO: 175, 177, or 181;

wherein the first primer or/and the second primer is optionally labeled with a labeling substance, which is bound via a covalent bond or a linker; and

wherein the first primer and the second primer are together capable of amplifying a genomic DNA fragment of Mycobacterium avium genome.

40 . The kit according to claim 39 , wherein the second primer of said primer pair is not more than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 176, 178, or 182.

41 . The reagent kit according to claim 39 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin.