Methods for rapid detection and identification of bacterial bioagents

Abstract

Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.

What is claimed is:  
     
         1 . A method of identifying an unknown bacterial bioagent comprising: 
 a) contacting nucleic acid from the bioagent with a pair of oligonucleotide primers which hybridize to sequences of the nucleic acid, wherein the sequences are between about 80-100% identical among different species of bioagents, wherein the sequences flank a variable nucleic acid sequence of the bioagent, and wherein the variable nucleic acid sequence exhibits no greater than about 5% identity among species and is between about 30 and 1000 nucleotides in length;    b) amplifying the variable nucleic acid sequence to produce a first amplification product;    c) determining the molecular mass or base composition of the first amplification product;    d) comparing the molecular mass or base composition of the first amplification product to one or more calculated or measured molecular masses or base compositions of amplification products of bacterial bioagents; and    e) performing steps a)-d) using at least one different oligonucleotide primer pair to obtain at least a second amplification product and comparing the results to one or more molecular masses or base compositions of amplification products of bacterial bioagents wherein at least one match determines the identity of the unknown bacterial bioagent.    
     
     
         2 . The method of  claim 1  wherein the amplifying step comprises polymerase chain reaction.  
     
     
         3 . The method of  claim 1  wherein the amplifying step comprises ligase chain reaction or strand displacement amplification.  
     
     
         4 . The method of  claim 1  wherein the nucleic acid encodes ribosomal RNA.  
     
     
         5 . The method of  claim 1  wherein the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.  
     
     
         6 . The method of  claim 1  wherein the amplification product is ionized prior to molecular mass determination.  
     
     
         7 . The method of  claim 1  further comprising the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the pair of oligonucleotide primers.  
     
     
         8 . The method of  claim 1  wherein the one or more molecular masses or base compositions are contained in a database.  
     
     
         9 . The method of  claim 1  wherein the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption, or fast atom bombardment.  
     
     
         10 . The method of  claim 1  wherein the molecular mass or base composition is determined by mass spectrometry.  
     
     
         11 . The method of  claim 10  wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.  
     
     
         12 . The method of  claim 1  further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.  
     
     
         13 . The method of  claim 1  wherein the oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within the primer, wherein the base analog binds with increased affinity to its complement compared to the native base.  
     
     
         14 . The method of  claim 13  wherein the primer comprises a universal base at position 3 of each triplet within the primer.  
     
     
         15 . The method of  claim 13  wherein the base analog is 2,6-diaminopurine, propyne T, propyne G, propyne C, phenoxazines, or G-clamp.  
     
     
         16 . The method of  claim 13  wherein the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.  
     
     
         17 . The method of  claim 1  wherein the sequences to which the pair of oligonucleotide primers hybridize are present within a gene involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion.  
     
     
         18 . The method of  claim 1  wherein the sequences to which oligonucleotide primers hybridize are between about 90-100% identical among different species of bioagents.  
     
     
         19 . The method of  claim 1  wherein the sequences to which oligonucleotide primers hybridize are between about 95-100% identical among different species of bioagents.  
     
     
         20 . A method of identifying an unknown bacterial bioagent comprising: 
 a) contacting nucleic acid from the bioagent with a pair of oligonucleotide primers which hybridize to sequences of the nucleic acid, wherein the sequences flank a variable nucleic acid sequence of the bioagent, and wherein the variable nucleic acid sequence exhibits no greater than about 5% identity among species and is between about 30 and 1000 nucleotides in length;    b) amplifying the variable nucleic acid sequence to produce a first amplification product;    c) determining the molecular mass or base composition of the first amplification product;    d) comparing the molecular mass or base composition of the first amplification product to one or more calculated or measured molecular masses or base compositions of amplification products of bacterial bioagents; and    e) performing steps a)-d) using at least one different oligonucleotide primer pair to obtain at least a second amplification product and comparing the results to one or more molecular masses or base compositions of amplification products of bacterial bioagents wherein at least one match determines the identity of the unknown bacterial bioagent.    
     
     
         21 . The method of  claim 20  wherein the amplifying step comprises polymerase chain reaction.  
     
     
         22 . The method of  claim 20  wherein the amplifying step comprises ligase chain reaction or strand displacement amplification.  
     
     
         23 . The method of  claim 20  wherein the nucleic acid encodes ribosomal RNA.  
     
     
         24 . The method of  claim 20  wherein the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.  
     
     
         25 . The method of  claim 20  wherein the amplification product is ionized prior to molecular mass determination.  
     
     
         26 . The method of  claim 20  further comprising the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the pair of oligonucleotide primers.  
     
     
         27 . The method of  claim 20  wherein the one or more molecular masses or base compositions are contained in a database.  
     
     
         28 . The method of  claim 20  wherein the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption, or fast atom bombardment.  
     
     
         29 . The method of  claim 20  wherein the molecular mass or base composition is determined by mass spectrometry.  
     
     
         30 . The method of  claim 29  wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.  
     
     
         31 . The method of  claim 20  further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.  
     
     
         32 . The method of  claim 20  wherein the oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within the primer, wherein the base analog binds with increased affinity to its complement compared to the native base.  
     
     
         33 . The method of  claim 32  wherein the primer comprises a universal base at position 3 of each triplet within the primer.  
     
     
         34 . The method of  claim 33  wherein the base analog is 2,6-diaminopurine, propyne T, propyne G, propyne C, phenoxazines, or G-clamp.  
     
     
         35 . The method of  claim 33  wherein the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.  
     
     
         36 . The method of  claim 20  wherein the sequences to which the pair of oligonucleotide primers hybridize are present within a gene involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion.  
     
     
         37 . A method of identifying an unknown bacterial bioagent comprising: 
 a) contacting nucleic acid from the bioagent with a pair of oligonucleotide primers which hybridize to sequences of the nucleic acid, wherein the sequences are between about 80-100% identical among different species of bioagents, and wherein the sequences flank a variable nucleic acid sequence of the bioagent which is between about 30 and 1000 nucleotides in length;    b) amplifying the variable nucleic acid sequence to produce a first amplification product;    c) determining the molecular mass or base composition of the first amplification product;    d) comparing the molecular mass or base composition of the first amplification product to one or more calculated or measured molecular masses or base compositions of amplification products of bacterial bioagents; and    e) performing steps a)-d) using at least one different oligonucleotide primer pair to obtain at least a second amplification product and comparing the results to one or more molecular masses or base compositions of amplification products of bacterial bioagents wherein at least one match determines the identity of the unknown bacterial bioagent    
     
     
         38 . The method of  claim 37  wherein the amplifying step comprises polymerase chain reaction.  
     
     
         39 . The method of  claim 37  wherein the amplifying step comprises ligase chain reaction or strand displacement amplification.  
     
     
         40 . The method of  claim 37  wherein the nucleic acid encodes ribosomal RNA.  
     
     
         41 . The method of  claim 37  wherein the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.  
     
     
         42 . The method of  claim 37  wherein the amplification product is ionized prior to molecular mass determination.  
     
     
         43 . The method of  claim 37  further comprising the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the pair of oligonucleotide primers.  
     
     
         44 . The method of  claim 37  wherein the one or more molecular masses or base compositions are contained in a database.  
     
     
         45 . The method of  claim 37  wherein the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption, or fast atom bombardment.  
     
     
         46 . The method of  claim 37  wherein the molecular mass or base composition is determined by mass spectrometry.  
     
     
         47 . The method of  claim 46  wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.  
     
     
         48 . The method of  claim 37  further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.  
     
     
         49 . The method of  claim 37  wherein the oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within the primer, wherein the base analog binds with increased affinity to its complement compared to the native base.  
     
     
         50 . The method of  claim 49  wherein the primer comprises a universal base at position 3 of each triplet within the primer.  
     
     
         51 . The method of  claim 49  wherein the base analog is 2,6-diaminopurine, propyne T, propyne G, propyne C, phenoxazines, or G-clamp.  
     
     
         52 . The method of  claim 50  wherein the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.  
     
     
         53 . The method of  claim 37  wherein the sequences to which the pair of oligonucleotide primers hybridize are present within a gene involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion.  
     
     
         54 . The method of  claim 37  wherein the sequences to which oligonucleotide primers hybridize are between about 90-100% identical among different species of bioagents.  
     
     
         55 . The method of  claim 37  wherein the sequences to which oligonucleotide primers hybridize are between about 95-100% identical among different species of bioagents.  
     
     
         56 . A method of identifying an unknown bacterial bioagent comprising: 
 a) contacting nucleic acid from the bioagent with a pair of oligonucleotide primers which hybridize to sequences of the nucleic acid, wherein the sequences flank a variable nucleic acid sequence of the bioagent that is between about 30 and 1000 nucleotides in length;    b) amplifying the variable nucleic acid sequence to produce a first amplification product;    c) determining the molecular mass of the first amplification product;    d) comparing the molecular mass of the first amplification product to one or more calculated or measured molecular masses of amplification products of bacterial bioagents; and    e) performing steps a)-d) using at least one different oligonucleotide primer pair to obtain at least a second amplification product and comparing the results to one or more molecular masses of amplification products of bacterial bioagents wherein at least one match determines the identity of the unknown bacterial bioagent.    
     
     
         57 . The method of  claim 56  wherein the amplifying step comprises polymerase chain reaction.  
     
     
         58 . The method of  claim 56  wherein the amplifying step comprises ligase chain reaction or strand displacement amplification.  
     
     
         59 . The method of  claim 56  wherein the nucleic acid encodes ribosomal RNA.  
     
     
         60 . The method of  claim 56  wherein the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.  
     
     
         61 . The method of  claim 56  wherein the amplification product is ionized prior to molecular mass determination.  
     
     
         62 . The method of  claim 56  further comprising the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the pair of oligonucleotide primers.  
     
     
         63 . The method of  claim 56  wherein the one or more molecular masses or base compositions are contained in a database.  
     
     
         64 . The method of  claim 56  wherein the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption, or fast atom bombardment.  
     
     
         65 . The method of  claim 56  wherein the molecular mass or base composition is determined by mass spectrometry.  
     
     
         66 . The method of  claim 65  wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, or triple quadrupole.  
     
     
         67 . The method of  claim 56  further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.  
     
     
         68 . The method of  claim 56  wherein the oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within the primer, wherein the base analog binds with increased affinity to its complement compared to the native base.  
     
     
         69 . The method of  claim 68  wherein the primer comprises a universal base at position 3 of each triplet within the primer.  
     
     
         70 . The method of  claim 68  wherein the base analog is 2,6-diaminopurine, propyne T, propyne G, propyne C, phenoxazines, or G-clamp.  
     
     
         71 . The method of  claim 68  wherein the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.  
     
     
         72 . The method of  claim 56  wherein the sequences to which the pair of oligonucleotide primers hybridize are present within a gene involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion.  
     
     
         73 . The method of  claim 56  wherein the variable nucleic acid sequence of the bioagent is no more than about 50-250 nucleotides.  
     
     
         74 . The method of  claim 56  wherein the variable nucleic acid sequence of the bioagent is no more than about 60-100 nucleotides.