Primer and probe for detection of mycobacterium avium and method for detection of mycobacterium avium by using the primer or probe

Abstract

The object of the present invention is to provide a novel primer for use in the detection of Mycobacterium avium ( M. avium ), and a method for detection of M. avium conveniently, rapidly and with high precision by using the primer. More specifically, disclosed are 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, and which is capable of hybridizing with the nucleotide sequence for a Mycobacterium avium gene; a primer and a probe for use in the detection of M. avium , which comprises the oligonucleoride; and a method for detection of M. avium using the primer and/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.