Primer and Probe for Use In Detection of Mycobacterium Kansasii and Method for Detection of Mycobacterium Kansasii Using The Same

Abstract

The present invention discloses an oligonucleotide which comprises a part or the entire sequence of the nucleotide sequence depicted in SEQ ID NOS: 1-4, or a part or the entire sequence of a nucleotide sequence complementary to SEQ ID NOS: 1-4, wherein the oligonucleotide is capable of hybridizing with the nucleotide sequence of Mycobacterium kansasii ; a primer and a probe for detecting M. kansasii comprising the oligonucleotide; and a method for detecting M. kansasii using the primer and/or probe. The methods enable the detection of M. kansasii more rapidly and with higher accuracy compared with conventional methods performed by culture. Further, the methods can exclude false positive results for the diagnosis and can also detect and diagnose M. kansasii with higher accuracy compared with methods performed by PCR using conventional primers and/or probes. Still further, the method can quantify M. kansasii cells.

1 - 29 . (canceled) 
     
     
         30 . A method for detecting  Mycobacterium kansasii  comprising:
 contacting a sample suspected of containing  Mycobacterium kansasii  with a primer comprising a first oligonucleotide consisting of 18 to 35 contiguous bases of a nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or 18 to 35 contiguous bases of the full complement of the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
 wherein the first oligonucleotide is capable of hybridizing with a nucleotide sequence of  Mycobacterium kansasii  gene. 
   
     
     
         31 . The method according to  claim 30 , wherein the first oligonucleotide consisting of 18 to 35 contiguous bases of the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or the full complement thereto consists of one of SEQ ID NOS: 5 to 52, or the full complement thereto. 
     
     
         32 . The method according to  claim 31 , wherein the first oligonucleotide consists of 18 to 35 contiguous bases of the nucleotide sequence consisting of SEQ ID NO: 1 or the full complement thereto, selected from one of SEQ ID NOS: 5 to 12 or the full complement thereto. 
     
     
         33 . The method according to  claim 31 , wherein the first oligonucleotide consists of 18 to 35 contiguous bases of the nucleotide sequence consisting of SEQ ID NO: 2 or the full complement thereto, selected from one of SEQ ID NOS: 13 to 26 or the full complement thereto. 
     
     
         34 . The method according to  claim 31 , wherein the first oligonucleotide consists of 18 to 35 contiguous bases of the nucleotide sequence consisting of SEQ ID NO: 3 or the full complement thereto, selected from one of SEQ ID NOS: 27 to 40 or the full complement thereto. 
     
     
         35 . The method according to  claim 31 , wherein the first oligonucleotide consists of 18 to 35 contiguous bases of the nucleotide sequence consisting of SEQ ID NO: 4 or the full complement thereto, selected from one of SEQ ID NOS: 41 to 52 or the full complement thereto. 
     
     
         36 . The method according to  claim 30 , further comprising;
 performing a nucleic acid amplification reaction using the primer to make a primer extension product; and   detecting the obtained primer extension product.   
     
     
         37 . The method according to  claim 36 , further comprising:
 performing the nucleic acid amplification reaction in the presence of a probe comprising a second oligonucleotide designed from a nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or from the full complement of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
 wherein the second oligonucleotide consists of 10 to 50 contiguous bases of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or 10 to 50 bases of the full complement of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; 
 wherein the probe is capable of hybridizing to the obtained primer extension product; and 
 wherein the probe and the primer hybridize to different positions of the  Mycobacterium kansasii  gene. 
   
     
     
         38 . The method according to  claim 37 , wherein the second oligonucleotide designed from the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or the full complement thereto consists of one of SEQ ID NOS: 53 to 80, or the full complement thereto. 
     
     
         39 . The method according to  claim 38 , wherein the probe is labeled with a labeling substance. 
     
     
         40 . The method according to  claim 39 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or biotin. 
     
     
         41 . The method according to  claim 40 , wherein the probe is labeled with a reporter fluorescent dye and with a quencher dye. 
     
     
         42 . The method according to  claim 41 , 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. 
     
     
         43 . The method according to  claim 30 , 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.   
     
     
         44 . The method according to  claim 43 , further comprising;
 identifying the obtained primer extension product in the obtained electrophoresis result based upon the expected length of the primer extension product.   
     
     
         45 . The method according to  claim 43 , 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 probe labeled with a labeling substance, wherein the probe comprises a second oligonucleotide designed from a nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or from the full complement of the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
 wherein the probe has a length of at least 10 to 700 nucleotides and at most a length up to the full length of SEQ ID NOS: 1 to 4 from which the probe is designed; and 
 wherein the second oligonucleotide is capable of hybridizing with a nucleotide sequence of  Mycobacterium kansasii  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. 
 
     
     
         46 . The method according to  claim 45 , wherein the probe comprises the second oligonucleotide designed from the nucleotide sequence consisting of:
 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the full complement of the nucleotide sequence consisting of SEQ ID NO; 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or   10 to 50 or 100 to 700 contiguous bases of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or 10 to 50 or 100 to 700 contiguous bases of the full complement of the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.   
     
     
         47 . The method according to  claim 46 , wherein the probe comprising the second oligonucleotide designed from the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or the full complement thereto consists of one of SEQ ID NOS: 5 to 80, or the full complement thereto. 
     
     
         48 . The method according to  claim 45 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or biotin. 
     
     
         49 . The method according to  claim 30 , 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.   
     
     
         50 . The method according to  claim 49 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or biotin. 
     
     
         51 . The method according to  claim 49 , further comprising after performing the nucleic acid amplification reaction;
 removing free labeled primer; and   measuring the signal derived from the labeled primer extension product.   
     
     
         52 . The method according to  claim 51 , wherein the free labeled primer is removed by removing a supernatant after precipitating the labeled primer extension product. 
     
     
         53 . The method according to  claim 51 , wherein free labeled primer is removed by gel chromatography. 
     
     
         54 . A method for detecting  Mycobacterium kansasii  comprising:
 contacting under hybridization conditions a sample suspected of containing  Mycobacterium kansasii  with a probe labeled with a labeling substance, wherein the probe comprises an oligonucleotide designed from a nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or from the full complement of the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;
 wherein the probe has a length of at least 10 to 700 nucleotides and at most a length up to the full length of SEQ ID NOS: 1 to 4 from which the probe is designed; and 
 wherein the oligonucleotide is capable of hybridizing with a nucleotide sequence of  Mycobacterium kansasii  gene. 
   
     
     
         55 . The method according to  claim 54 , wherein the probe comprises:
 the oligonucleotide consisting of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or the full complement of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or   the oligonucleotide consisting of 10 to 50 or 100 to 700 contiguous bases of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or 10 to 50 or 100 to 700 contiguous bases of the full complement of the sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.   
     
     
         56 . The method according to  claim 55 , wherein the oligonucleotide designed from the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or the full complement thereto consists of one of SEQ ID NOS: 5 to 80, or the full complement thereto. 
     
     
         57 . The method according to  claim 55 , wherein the oligonucleotide designed from the nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or the full complement thereto consists of one of SEQ ID NOS: 53 to 79, or the full complement thereto. 
     
     
         58 . The method according to  claim 54 , wherein the labeling substance is selected from a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or biotin. 
     
     
         59 . The method according to  claim 54 , further comprising:
 separating free labeled probe from the hybridized complex; and   detecting a signal derived from the labeled probe associated with the hybridized complex.