Method for rapid detection and identification of bioagents

• US 8,017,358 B2
• Filed: 01/13/2006
• Issued: 09/13/2011
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Hofstadler, Steven, Sampath, Rangarajan, Griffey, Richard H., McNeil, John, Ecker, David J.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Pande; Suchira
• Application Number:
  US11/331,987
• Publication Number:
  US 20060121520A1
• Time in Patent Office: 2,069 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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.

Claims

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.