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 oligonucleotide; and a method for detection of M. avium using the primer and/or probe.

1 - 21 . (canceled) 
     
     
         22 . 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 selected from SEQ ID NO: 165, 167, and 169;   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.   
     
     
         23 . The primer pair 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 pair according to  claim 22 , wherein the second primer of said primer pair is not more than about 50 nucleotides in length and comprises the nucleotide sequence selected from SEQ ID NO: 166, 168, and 170. 
     
     
         25 . A reagent kit comprising:
 (i) a primer set consisting of   (a) a first primer consisting of a primer that is not more than about 50 nucleotides in length and comprises an oligonucleotide that comprises the nucleotide sequence selected from SEQ ID NO: 165, 167, and 169, is labeled with a labeling substance, which is bound via a covalent bond or a linker, and is capable of hybridizing with a genomic DNA fragment of  Mycobacterium avium  genome; and   (b) optionally, an additional primer, wherein the additional primer is not more than about 50 nucleotides in length and comprises a contiguous portion of at least 15 nucleotides of the complementary sequence of SEQ ID NO: 134, and is capable of hybridizing with the 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.   
     
     
         26 . The reagent kit according to  claim 25 , further comprising:
 a probe, wherein the probe comprises (a) an oligonucleotide that comprises a nucleotide sequence selected from:   SEQ ID NO: 134, or the full nucleotide sequence complementary thereto, wherein the probe is no longer than about 700 nucleotides; or   SEQ ID NO: 165 to 170, or the full nucleotide sequence complementary thereto, wherein the probe is no longer than about 50 nucleotides; or   SEQ ID NO: 197 to 199, or the full nucleotide sequence complementary thereto; or   20-50 consecutive nucleotides of SEQ ID NO: 197 to 199, or the 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.   
     
     
         27 . A reagent kit comprising:
 (i) a primer pair consisting of a first primer and a second primer, wherein (a) the first primer is not more than about 50 nucleotides in length and comprises an oligonucleotide that comprises the nucleotide sequence selected from SEQ ID NO: 165, 167, and 170; (b) 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 (c) the first primer and the second primer are 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.   
     
     
         28 . The reagent kit according to  claim 27 , further comprising:
 a probe, wherein the probe comprises (a) an oligonucleotide that comprises a nucleotide sequence selected from:   SEQ ID NO: 134, or the full nucleotide sequence complementary thereto, wherein the probe is no longer than about 700 nucleotides; or   SEQ ID NO: 165 to 170, or the full nucleotide sequence complementary thereto, wherein the probe is no longer than about 50 nucleotides; or   SEQ ID NO: 197 to 199, or the full nucleotide sequence complementary thereto; or   20-50 consecutive nucleotides of SEQ ID NO: 197 to 199, or the full nucleotide sequence complementary thereto,   wherein the probe is capable of hybridizing with a genomic DNA fragment of  Mycobacterium avium  genome; and   optionally (b) a labeling substance, which is bound to the oligonucleotide via a covalent bond or a linker.   
     
     
         29 . The reagent kit according to  claim 27 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin. 
     
     
         30 . The reagent kit according to  claim 27 , wherein the second primer of said primer pair is not more than about 50 nucleotides in length and comprises the nucleotide sequence selected from SEQ ID NO: 166, 168, and 170. 
     
     
         31 . A reagent kit comprising:
 (i) a primer composition consisting essentially of a first primer or a primer pair,   wherein the first primer is not more than about 50 nucleotides in length and comprises an oligonucleotide that comprises the nucleotide sequence selected from SEQ ID NO: 165, 167, and 169;   wherein the first primer is capable of hybridizing with a genomic DNA fragment of  Mycobacterium avium  genome; and   wherein the primer pair consists of the first primer and a second primer, and the first primer and the second primer are capable of amplifying a genomic DNA fragment of  Mycobacterium avium  genome; and   (ii) a probe consisting of   (a) an oligonucleotide that comprises a nucleotide sequence selected from:   SEQ ID NO: 134, or the full length sequence complementary thereto, wherein the probe is no longer than about 700 nucleotides; or   SEQ ID NO: 165 to 170, or the full nucleotide sequence complementary thereto, wherein the probe is no longer than about 50 nucleotides; or   SEQ ID NO: 197 to 199, or the full nucleotide sequence complementary thereto; or   20-50 consecutive nucleotides of SEQ ID NO: 197 to 100, or the full nucleotide sequence complementary thereto;   wherein the probe is capable of hybridizing with a genomic DNA fragment of  Mycobacterium avium  genome, and   (b) a labeling substance bound to the oligonucleotide via a covalent bond or a linker; wherein the labeling substance comprises a reporter fluorescent dye and a quencher dye; and   (iii) (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.   
     
     
         32 . The reagent kit according to  claim 31 , wherein the reporter fluorescent dye is covalently bound to the 5′-terminal of the oligonucleotide and the quencher dye is covalently bound to the 3′-terminal of the oligonucleotide.