Ribosomal Ribonucleic Acid Hybridization for Organism Identification

Abstract

The present disclosure relates to method of distinguishing between two or more species of one or more organisms in a sample, by contacting a biological sample comprising ribosomal ribonucleic acid (rRNA) with a set of antisense probes, wherein the set of probes contains at least one detectable probe that is specific for a target rRNA sequence of each species to be tested, and wherein the individual probes specific for each species comprises less than about 85% sequence identity; and, detecting hybridization between one or more of the probes and the rRNA, thereby distinguishing between two or more species in a sample.

What is claimed is: 
     
         1 . A method of distinguishing between two or more species of one or more organisms in a sample, comprising:
 contacting a sample comprising ribosomal ribonucleic acid (rRNA) with a set of antisense probes, wherein the set of probes contains at least one detectable probe that is specific for a target rRNA sequence of each species to be tested, and wherein the individual probes specific for each species have about 85% or less sequence identity to the probes for the other species; and,   detecting hybridization between one or more of the probes and the rRNA, thereby distinguishing between two or more species in a sample.   
     
     
         2 . The method of  claim 1 , further comprising determining the at least one detectable probe that is specific for a target rRNA sequence of each species, wherein the determination comprises:
 determining shared rRNA sequences between members of each of the two or more species;   determining divergent rRNA sequences between each of the two or more species, wherein the divergent rRNA sequences share 85% or less sequence identity, to identify a target RNA sequence for each species; and   designing a probe for the target rRNA sequence of each species that specifically binds to the shared rRNA sequences between members of a given species, wherein the individual probes for are each species are about 85% or less identical.   
     
     
         3 . The method of  claim 1 , wherein the one or more organisms comprise one or more microorganism. 
     
     
         4 . The method of  claim 2 , wherein the one or more microorganism is selected from the group consisting of bacteria, yeast, and fungi or a combination thereof. 
     
     
         5 . The method of  claim 3  or  4 , wherein the one or more microorganism is a human pathogen. 
     
     
         6 . The method of any one of  claims 3 - 5 , wherein the one or more microorganism comprises bacteria. 
     
     
         7 . The method of  claim 4 - 5 , wherein the one or more microorganism comprises one or more of  Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Streptococcus agalactiae , or  Stenotrophomonas maltophilia.    
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the target rRNA sequence of each species to be tested comprise at least 2 of the nucleic acid sequences set forth as SEQ ID NO: 107-159, and wherein each species specific probe in set of antisense probes specifically binds to the nucleic acid sequences set forth as SEQ ID NOs: 107-159. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the set of antisense probes comprises one or more probes to  Escherichia coli , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the set of antisense probes comprises one or more probes to  Klebsiella pneumoniae , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77, or SEQ ID NO: 78. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the set of antisense probes comprises one or more probes to  Pseudomonas aeruginosa , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86, or SEQ ID NO: 87. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the set of antisense probes comprises one or more probes to  Staphylococcus aureus , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the set of antisense probes comprises one or more probes to  Staphylococcus maltophilia , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 40, or SEQ ID NO: 41, SEQ ID NO: 93, or SEQ ID NO: 94. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the set of antisense probes comprises one or more probes to  Staphylococcus agalactiae , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 37, or SEQ ID NO: 38, SEQ ID NO: 90, or SEQ ID NO: 91. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the set of antisense probes comprises one or more probes to  Staphylococcus  sp, and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 88, or SEQ ID NO: 89. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the set of antisense probes comprises one or more probes to  Proteus mirabilis , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 27, or SEQ ID NO: 80. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the set of antisense probes comprises one or more probes to  Enterococcus faecium , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 62, or SEQ ID NO: 63. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the set of antisense probes comprises one or more probes to  Enterococcus faecalis , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 8 or SEQ ID NO: 61. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the set of antisense probes comprises one or more probes to  Acinetobacter baumanniii , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 54, or SEQ ID NO: 55. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the set of antisense probes comprises one or more probes to  Candida albicans , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 56, or SEQ ID NO: 57. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the set of antisense probes comprises one or more probes to  Enterobacter cloacae , and wherein the one or more probes is at least 95% identical to the nucleic acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 58. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the sample is a biological sample obtained from a subject. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein the detecting hybridization between the probe indicates the presence of the species in the sample. 
     
     
         24 . The method of  claim 23 , wherein the presence of the species in the sample indicates a subject from which the sample was obtained is infected with the organism. 
     
     
         25 . The method of any one of  claims 1 - 24 , wherein the detectable probe is labeled. 
     
     
         26 . The method of  claim 25 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         27 . The method of  claim 26 , wherein the label comprises a capture moiety. 
     
     
         28 . The method of  claim 27 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         29 . The method of any one of  claims 1 - 28 , wherein the probe is attached to a solid surface. 
     
     
         30 . The method of any one of  claims 1 - 29 , further comprising contacting the sample with a second set of antisense probes, wherein the second set of probes contains at least one second detectable probe that is specific for each species to be tested, and wherein the individual probes bind to substantially the same region of the rRNA as the first set of probes, but wherein the second set of probes do not overlap in sequence identity. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein at least one second detectable probe that is specific for each species to be tested specifically bind the nucleic acid sequences set forth as one of SEQ ID NOs: 107-159. 
     
     
         32 . The method of  claim 30  or  31 , wherein the second detectable probe is labeled. 
     
     
         33 . The method of  claim 32 , wherein the second detectable probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         34 . The method of  claim 32  or  33 , wherein the label comprises a capture moiety. 
     
     
         35 . The method of  claim 34 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         36 . The method of any one of claim  2126 , wherein the second detectable probe is attached to a solid surface. 
     
     
         37 . A method for detecting a bacterial species in a sample, comprising:
 contacting the sample with one or more detectable probes comprising a nucleic acid sequence capable of hybridizing to a bacterial ribosomal ribonucleic acid sequence set forth as one of SEQ ID NOs: 106-159, and   detecting hybridization between the bacterial ribosomal ribonucleic acid and the probe, wherein the detection of the hybridization indicates the bacterial species is present.   
     
     
         38 . The method of  claim 37 , wherein the one or more detectable probes comprises the nucleic acid sequence set forth as SEQ ID NO: 1-106. 
     
     
         39 . The method of  claim 37  or  38 , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71 indicates the presence of  Escherichia coli , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77, or SEQ ID NO: 78 indicates the presence of  Klebsiella pneumoniae , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86, or SEQ ID NO: 87 indicates the presence of  Pseudomonas aeruginosa , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 indicates the presence of  Staphylococcus aureus , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 56, or SEQ ID NO: 57 indicates the presence of  Candida albicans , wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 58 indicates the presence of  Enterobacter cloacae  wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 8 or SEQ ID NO: 61 indicates the presence of  Enterococcus faecalis  wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 62, or SEQ ID NO: 63 indicates the presence of  Enterococcus faecium  wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 27, or SEQ ID NO: 80 indicates the presence of  Proteus mirabilis  wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 37, or SEQ ID NO: 38, SEQ ID NO: 90, or SEQ ID NO: 91 indicates the presence of  Staphylococcus agalactiae  wherein detection of a bacterial ribosomal ribonucleic acid sequence with one or more probes at least 95% identical to SEQ ID NO: 40, or SEQ ID NO: 41, SEQ ID NO: 93, or SEQ ID NO: 94 indicates the presence of  Staphylococcus maltophilia.    
     
     
         40 . The method of any one of  claims 37 - 39 , further comprising discriminating between an a  Klebsiella pneumoniae  ribosomal ribonucleic acid, a  Pseudomonas aeruginosa  ribosomal ribonucleic acid, a  Staphylococcus aureus  ribosomal ribonucleic acid an  Acinetobacter baumannii  ribosomal ribonucleic acid, a  Candida albicans  ribosomal ribonucleic acid, a  Enterobacter cloacae  ribosomal ribonucleic acid, a  Enterococcus faecalis  ribosomal ribonucleic acid, a  Enterococcus faecium  ribosomal ribonucleic acid, a  Proteus mirabilis  ribosomal ribonucleic acid, or a  Staphylococcus  sp. ribosomal ribonucleic acid, a  S. agalactiae  ribosomal ribonucleic acid, or a  S. maltophilia  ribosomal ribonucleic acid, the method further comprising:
 contacting the sample with a probe specific for the  Klebsiella pneumoniae  ribosomal ribonucleic acid, the  Pseudomonas aeruginosa  ribosomal ribonucleic acid, the  Staphylococcus aureus  ribosomal ribonucleic acid the  Acinetobacter baumannii  ribosomal ribonucleic acid, the  Candida albicans  ribosomal ribonucleic acid, the  Enterobacter cloacae  ribosomal ribonucleic acid, the  Enterococcus faecalis  ribosomal ribonucleic acid, the  Enterococcus faecium  ribosomal ribonucleic acid, the  Proteus mirabilis  ribosomal ribonucleic acid, the  S. agalactiae  ribosomal ribonucleic acid, or the  S. maltophilia  ribosomal ribonucleic acid; and,   detecting hybridization between the probe and the  Pseudomonas aeruginosa  ribosomal ribonucleic acid, the  Staphylococcus aureus  ribosomal ribonucleic acid the  Acinetobacter baumannii  ribosomal ribonucleic acid, the  Candida albicans  ribosomal ribonucleic acid, the  Enterobacter cloacae  ribosomal ribonucleic acid, the  Enterococcus faecalis  ribosomal ribonucleic acid, the  Enterococcus faecium  ribosomal ribonucleic acid, the  Proteus mirabilis  ribosomal ribonucleic acid, the  S. ag.      
     
     
         41 . The method of any of  claims 37 - 40 , wherein the sample is a biological sample from a subject. 
     
     
         42 . The method of  claim 41 , wherein the presence of the bacterial species in the biological sample from the subject indicates the presence of the bacterial species in the subject. 
     
     
         43 . The method of any one of  claims 37 - 42 , wherein the detectable probe is labeled. 
     
     
         44 . The method of any one of  claims 37 - 43 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         45 . The method of  claim 44 , wherein the label comprises a capture moiety. 
     
     
         46 . The method of  claim 45 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         47 . The method of any one of  claims 37 - 46 , wherein the probe is attached to a solid surface. 
     
     
         48 . A method for classifying a bacterial strain in a sample, comprising:
 contacting the sample with one or more detectable antisense ribonucleic acid probes comprising a ribonucleic acid sequence capable of hybridizing to a bacterial ribosomal ribonucleic acid sequence, the one or more probes comprising the sequence set forth as SEQ ID NO: 1-106; and   detecting hybridization between the bacterial ribosomal ribonucleic acid and the at one or more detectable probe, wherein the detection of the hybridization indicates the classification of the bacterial strain.   
     
     
         49 . The method of  claim 48 , wherein the sample is a biological sample from a subject. 
     
     
         50 . The method of any one of  claim 48  or  49 , wherein the detectable probe is labeled. 
     
     
         51 . The method of any one of  claims 48 - 50 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         52 . The method of  claim 51 , wherein the label comprises a capture moiety. 
     
     
         53 . The method of  claim 52 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         54 . The method of any one of  claims 48 - 53 , wherein the probe is attached to a solid surface. 
     
     
         55 . A ribosomal ribonucleic acid (rRNA) probe for the detection of a bacterial species, comprising a nucleic acid sequence between 25 and 120 nucleotides in length capable of hybridizing to a bacterial species rRNA sequence, the probe comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth as set forth as one of SEQ ID NO: 1-106, wherein the probe is labeled. 
     
     
         56 . The probe according  claim 55 , wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. 
     
     
         57 . The probe according to  claim 56 , wherein the label comprises a capture moiety. 
     
     
         58 . The probe according to  claim 57 , wherein the capture moiety comprises biotin, linked to a nucleotide. 
     
     
         59 . A set of probes comprising at least on probe of any one of  claims 55 - 58 . 
     
     
         60 . A kit for detecting a bacterial ribosomal ribonucleic acid in a sample, comprising:
 one or more probes of any of  claims 55 - 58 ; and,   instructions for hybridizing the probe to a bacterial ribosomal ribonucleic acid in the sample.   
     
     
         61 . A device for detecting one or more microorganism is a sample, the device comprising one or more probes of any of  claims 55 - 58  linked to a solid surface. 
     
     
         62 . A method of diagnosing a species-specific bacterial infection in a subject suspected of having a bacterial infection, comprising:
 obtaining a sample comprising bacterial ribosomal ribonucleic acid from the subject;   contacting the sample with one or more of the probes according to  claims 55 - 58 ,   detecting hybridization between the bacterial ribosomal ribonucleic acid sequence present in the sample and the probe, wherein the detection of hybridization indicates that the subject is infected with a bacteria.