Method for rapid detection and identification of bioagents

• US 20060121520A1
• Filed: 01/13/2006
• Published: 06/08/2006
• Est. Priority Date: 03/02/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Sampath, Rangarajan, McNeil, John, Ecker, David J., Hofstadler, Steven, Griffey, Richard
• Granted Patent: US 8,017,358 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 designing a pair of primers for use in bioagent identification 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; and

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.

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 universal base.

5. The method of claim 4 wherein said universal base is inosine.

6. The method of claim 1 wherein said pair of primers comprises at least one modified nucleobase.

7. The method of claim 6 wherein said modified nucleobase comprises a propynylated pyrimidine.

8. The method of claim 1 wherein said gene sequence encodes a protein involved in a core life function.

9. The method of claim 1 wherein said gene sequence encodes ribosomal RNA.

10. The method of claim 1 wherein said primer binding sites are separated by between about 60 to 100 nucleobases.

11. A primer pair designed according to the method of claim 1.

12. An amplification product produced by a process of amplifying nucleic acid of a bioagent genome with the primer pair of claim 11.

13. A method of identifying a bioagent comprising:

obtaining one or more amplification products by amplification of nucleic acid of said bioagent with one or more primer pairs designed according to the method of claim 1;

measuring the base composition of said one or more amplification products;

comparing said base composition with a plurality of base compositions of amplification products of nucleic acid of bioagents produced with said one or more primer pairs wherein one or more matches of said base composition with said plurality of base compositions identifies said bioagent.

14. A method of designing a pair of primer sequences for use in bioagent identification 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 wherein said variability is defined by no more than 5% sequence identity among said plurality of bioagent genomes which is flanked by upstream and downstream conserved regions of 80% to 100% sequence identity among said plurality of bioagent genomes;

identifying said conserved regions of 80% to 100% sequence identity as primer binding sites; and

designing a pair of primer sequences such that each member of said pair is hybridizable to one or the other of said conserved regions.

15. The method of claim 14 wherein said plurality of genomes comprises 19 or more genomes.

16. The method of claim 14 wherein said plurality of genomes comprises one hundred or more genomes.

17. The method of claim 14 wherein said gene region is between about 46 and 166 nucleobases in length.

18. The method of claim 14 wherein said primer binding sites are separated by between about 60 to 100 nucleobases.

19. A primer pair designed according to the method of claim 14.

20. An amplification product produced by the process of amplifying nucleic acid of a bioagent genome with the primer pair of claim 19.

21. A method of identifying a bioagent comprising:

obtaining one or more amplification products by amplification of bioagent nucleic acid with one or more primer pairs of claim 19;

measuring the base composition of said one or more amplification products;

comparing said base composition with a plurality of base compositions of amplification products of nucleic acid of known bioagents produced with said one or more primer pairs wherein one or more matches of said base composition with said plurality of base compositions identifies said bioagent.