Method for rapid detection and identification of bioagents
View Patent ↗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.
1. A method of identifying an unknown bioagent by producing an ionized bioagent characterizing amplification product comprising:
aligning a gene sequence from a plurality of bioagent genomes to form an alignment;
selecting a gene region from said alignment comprising a region of variability defined by no more than 5% sequence identity among said plurality of bioagent genomes, which is flanked by a first and a second conserved region, each having 80% to 100% sequence identity among said plurality of bioagent genomes;
identifying said first and second conserved regions as primer binding sites;
designing a pair of primers comprising a first primer hybridizable to said first conserved region and second primer hybridizable to said second conserved region such that a plurality of amplification products of lengths of about 46 to 166 nucleobases can be produced upon amplification of said gene region with said pair of primers, wherein at least 8 of said plurality of amplification products have unique base compositions, wherein said base compositions that are present in a database identify the number, but not the nucleic acid gene sequence order, of A residues, C residues, T residues, G residues, U residues, analogues thereof and mass tag residues thereof in said amplification products;
amplifying a nucleic acid from an unknown bioagent with said pair of primers to produce a bioagent characterizing amplification product;
ionizing said amplification product;
determining the base composition of said ionized amplification product;
comparing said base composition of said ionized amplification product with said database containing said base compositions of said plurality of amplification products; and
identifying said unknown bioagent using said base composition of said ionized amplification product without sequencing said ionized amplification product.
2. The method of claim 1 wherein said plurality of bioagent genomes comprises 19 or more genomes.
3. The method of claim 1 wherein said plurality of bioagent genomes comprises one hundred or more genomes.
4. The method of claim 1 wherein said pair of primers comprises at least one modified nucleobase.
5. The method of claim 1 wherein said gene sequence encodes a protein molecule involved in a core life function.
6. The method of claim 1 wherein said gene sequence encodes ribosomal RNA.
7. The method of claim 1 wherein said primer binding sites are separated by between about 60 to 100 nucleobases.