Primer And Probe For Detection Of Mycobacterium Intracellulare, And Method For Detection Of Mycobacterium Intracellulare Using The Same

Abstract

The present invention discloses an oligonucleotide which comprises a part or the entire sequence of the nucleotide sequence shown in SEQ ID NOS: 1-8, or a part or the entire sequence of a sequence complementary to the nucleotide sequence shown in SEQ ID NOS: 1-8, wherein the oligonucleotide is capable of hybridizing with a nucleotide sequence of Mycobacterium intracellulare gene; a primer or a probe for the detection of M. intracellulare , which comprises the oligonucleotide; and a method for detection of M. intracellulare using the primer and/or the probe. According to the detection method, false-positive results can be eliminated and detection of M. intracellulare can be carried out with higher accuracy, greater precision, and greater specificity compared to a conventional diagnostic method employing a cell culture assay or a PCR assay. The method also enables to quantify a microbial cell.

1 - 21 . (canceled) 
     
     
         22 . A method for detecting  Mycobacterium intracellulare  in a sample, comprising:
 contacting a primer comprising an oligonucleotide designed from a nucleotide sequence selected from SEQ ID NOS: 1 to 8, or the sequence fully complementary thereto, wherein the primer has a length of 18 to 25 nucleotides, with a sample suspecting of containing a  Mycobacterium intracellulare  gene.   
     
     
         23 . The method according to  claim 22 , wherein the primer comprises an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 9-138, or the nucleotide sequence fully complementary thereto. 
     
     
         24 . The method according to  claim 22 , wherein the primer comprises an oligonucleotide designed from SEQ ID NO: 1 consisting of the nucleotide sequence of SEQ ID NOS: 9-22, or the nucleotide sequence fully complementary thereto. 
     
     
         25 . The method according to  claim 22 , further comprising;
 performing a nucleic acid amplification reaction using the primer to make a primer extension product; and   detecting the obtained primer extension product.   
     
     
         26 . The method according to  claim 25 , wherein the nucleic acid amplification reaction is selected from polymerase chain reaction, loop-mediated isothermal amplification, isothermal and chimeric primer-initiated amplification of nucleic acids, and strand displacement amplification. 
     
     
         27 . The method according to  claim 26 , further comprising:
 performing the nucleic acid amplification reaction in the presence of a probe comprising an oligonucleotide designed from a nucleotide sequence selected from SEQ ID NOS: 1 to 8, or the sequence fully complementary thereto, wherein the oligonucleotide is 10 to 50 nucleotides in length, wherein the probe is capable of hybridizing to the obtained primer extension product, further wherein the probe and the primer hybridize to different positions of the  Mycobacterium intracellulare  gene.   
     
     
         28 . The method according to  claim 27 , wherein the probe is labeled with a labeling substance. 
     
     
         29 . The method according to  claim 28 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and biotin. 
     
     
         30 . The method according to  claim 27 , wherein the probe is labeled with a reporter fluorescent dye and with a quencher dye. 
     
     
         31 . The method according to  claim 30 , wherein the 5′-terminal of the probe is labeled with the reporter fluorescent dye and the 3′-terminal of the probe is labeled with the quencher dye. 
     
     
         32 . The method according to  claim 22 , further comprising:
 performing a nucleic acid amplification reaction using the primer to make a primer extension product; and   separating the obtained primer extension product by electrophoresis.   
     
     
         33 . The method according to  claim 32 , further comprising:
 identifying the obtained primer extension product in the obtained electrophoresis result based upon the expected length of the primer extension product.   
     
     
         34 . The method according to  claim 32 , further comprising:
 obtaining an electrophoretic fraction containing the obtained primer extension product from the performed electrophoresis;   contacting under hybridization conditions the electrophoretic fraction with a labeled probe prepared by labeling an oligonucleotide designed from the nucleotide sequence selected from SEQ ID NOS: 1 to 8, or the sequence fully complementary thereto with a labeling substance, wherein the probe has a length of at least 10 to 700 nucleotides and at most a length of up to the full length of SEQ ID NOS: 1 to 8 from which the probe is designed, and wherein the labeled probe is capable of hybridizing with the nucleotide sequence of a  Mycobacterium intracellulare  gene, to form a hybridized complex between the primer extension product and the labeled probe; and   detecting a signal derived from the labeled probe associated with the hybridized complex.   
     
     
         35 . The method according to  claim 34 , wherein the probe comprises:
 an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 1 to 8, or the sequence fully complementary thereto;   an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 9 to 138, or 204, or the sequence fully complementary thereto, wherein the probe is no more than about 50 nucleotides in length; or   an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 139 to 203, or the sequence fully complementary thereto, wherein the probe is no more than about 500 nucleotides in length   
     
     
         36 . The method according to  claim 22 , wherein the primer is labeled with a labeling substance; and the method further comprises:
 performing a nucleic acid amplification reaction using the labeled primer to make a labeled primer extension product; and   measuring a signal derived from the labeled primer extension product.   
     
     
         37 . The method according to  claim 36 , further comprising separating from the labeled primer extension product any free labeled primer, and measuring the signal derived from the labeled primer extension product. 
     
     
         38 . A method for detecting  Mycobacterium intracellulare  in a sample, comprising:
 contacting a labeled probe prepared by labeling an oligonucleotide designed from a nucleotide sequence selected from SEQ ID NO: 1 to 8, or the sequence fully complementary thereto, wherein the probe has a length of at least 10 to 700 nucleotides and at most a length of up to the full length of SEQ ID NOS: 1 to 8 from which the probe is designed, with a labeling substance, wherein the labeled probe is capable of hybridizing with a nucleotide sequence of a  Mycobacterium intracellulare  gene, with a sample suspecting of containing a  Mycobacterium intracellulare  gene under hybridization conditions to form a hybridized complex;   separating from the hybridized complex any free labeled probe; and   detecting a signal derived from the labeled probe associated with the hybridized complex.   
     
     
         39 . The method according to  claim 38 , wherein the probe comprises
 an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 1 to 8, or the sequence fully complementary thereto;   an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 9 to 138, or 204, or the sequence fully complementary thereto, wherein the probe is no more than about 50 nucleotides in length; or   an oligonucleotide consisting of the nucleotide sequence of SEQ ID NOS: 139 to 203, or the sequence fully complementary thereto, wherein the probe is no more than about 500 nucleotides in length.