Methods for identifying bioagents

• US 8,563,250 B2
• Filed: 03/05/2007
• Issued: 10/22/2013
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Griffey, Richard H., Sampath, Rangarajan, Hofstadler, Steven, McNeil, John, Crooke, Stanley T., Hannis, James C.
• Primary Examiner(s): Woolwine, Samuel
• Application Number:
  US11/682,259
• Publication Number:
  US 20120122086A1
• Time in Patent Office: 2,423 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The present invention provides methods for rapid forensic investigations by identification of bioagents associated with biowarfare and acts of terrorism or crime. The methods are also useful for epidemiological investigations by genotyping of bioagents.

Claims

1. A method of identifying a bioagent in a sample comprising:

a) selecting at least three oligonucleotide primer pairs, wherein each of said oligonucleotide primer pairs is configured to hybridize at conserved primer binding sequences flanking a variable sequence within a gene selected from the group consisting of a sequence encoding a 16S ribosomal RNA, a 23S ribosomal RNA, sspE, and RNase P wherein said variable sequence is sufficiently variable to resolve the identity of said bioagent;

b) obtaining a sample comprising at least one bioagent;

c) amplifying nucleic acid from said at least one bioagent with said at least three oligonucleotide primer pairs to produce at least three different amplicons;

d) analyzing said amplicons to identify one or more bioagents in said sample wherein said analyzing further comprises generating molecular mass data for said amplicons; and

e) calculating a base composition from said generated molecular mass data.

2. The method of claim 1 wherein said conserved primer binding sequences from said 16S gene sequence have at least 80% identity with sequences selected from the group consisting of:

1100-1188 of SEQ ID NO. 3;

971-1062 of SEQ ID NO. 3;

714-805 of SEQ ID NO. 3; and

787-894 of SEQ ID NO. 3.

3. The method of claim 1 wherein said conserved primer binding sequences from said 23S gene sequence have at least 80% identity with sequences selected from the group consisting of:

2601-2667 of SEQ ID NO. 4; and

1840-1903 of SEQ ID NO. 4.

4. The method of claim 1 wherein a first oligonucleotide primer pair is configured to hybridize with a sequence from a 16S gene, a second oligonucleotide primer pair is configured to hybridize with a sequence from a 23S gene, and a third oligonucleotide primer pair is configured to hybridize with an additional sequence from a 23S gene.

5. The method of claim 1 wherein said at least three oligonucleotide primer pairs are configured to hybridize with three different segments from a 16S gene.

6. The method of claim 1 further comprising comparing said molecular mass data to a plurality of molecular masses in a database, wherein a match between said generated molecular masses and a member of said plurality of molecular masses identifies the genus, species, subspecies, serotype or genotype of said one or more bioagents.

7. The method of claim 1 further comprising comparing said base composition to a plurality of base compositions in a database, wherein a match between said base composition and a member of said plurality of base compositions identifies the genus, species, sub-species, serotype or genotype of said one or more bioagents.

8. The method of claim 1 wherein at least two of said amplicons differ in length.

9. The method of claim 1 wherein at least two of said amplicons differ in base composition.

10. The method of claim 1 wherein at least two of said amplicons differ in molecular mass.

11. The method of claim 1 wherein said amplicons are produced from different binding sites within the same gene.

12. The method of claim 1 wherein said amplicons are produced from different binding sites within different genes.

13. A method of generating bioagent identifying amplicons comprising the steps of:

a) selecting at least three oligonucleotide primer pairs, wherein each of said oligonucleotide primer pairs hybridize with conserved primer binding sequences having at least 80% identity with sequences flanking a variable sequence of a gene sequence selected from the group consisting of sequences 1100-1188 of SEQ ID NO. 3;

971-1062 of SEQ ID NO. 3;

714-805 of SEQ ID NO. 3; and

787-894 of SEQ ID NO. 3;

2601-2667 of SEQ ID NO. 4; and

1840-1903 of SEQ ID NO. 4 wherein said variable sequence is sufficiently variable to resolve the identity of said bioagent;

b) obtaining a sample comprising at least one bioagent;

c) amplifying nucleic acid from said at least one bioagent with said at least three oligonucleotide primer pairs to produce amplicons; and

d) analyzing said amplicons to identify one or more bioagents in said sample wherein said analyzing comprises mass spectrometry analysis.

14. The method of claim 13 wherein said mass spectrometry analysis is ESI TOF mass spectrometry.

15. The method of claim 13 wherein said analyzing comprises generating molecular mass data for said amplicons.

16. The method of claim 15 wherein said analyzing further comprises calculating a base composition from said generated molecular mass data.

17. The method of claim 16 further comprising comparing said base composition to a plurality of base compositions in a database, wherein a match between said base composition and a member of said plurality of base compositions identifies the genus, species, sub-species, serotype or genotype of said one or more bioagents.

18. The method of claim 15 further comprising comparing said molecular mass data to a plurality of molecular masses in a database, wherein a match between said generated molecular masses and a member of said plurality of molecular masses identifies the genus, species, subspecies, serotype or genotype of said one or more bioagents.

19. A method of identifying a bioagent in a sample comprising:

a) selecting at least three oligonucleotide primer pairs, wherein each of said oligonucleotide primer pairs is configured to hybridize at conserved primer binding sequences flanking a variable sequence within a gene selected from the group consisting of a sequence encoding a 16S ribosomal RNA, a 23S ribosomal RNA, sspE, and RNase P wherein said variable sequence is sufficiently variable to resolve the identity of said bioagent;

b) obtaining a sample comprising at least one bioagent;

c) amplifying nucleic acid from said at least one bioagent with said at least three oligonucleotide primer pairs to produce at least three different amplicons;

d) analyzing said amplicons to identify one or more bioagents in said sample wherein said analyzing further comprises generating molecular mass data for said amplicons; and

e) comparing said molecular mass data to a plurality of molecular masses in a database, wherein a match between said generated molecular masses and a member of said plurality of molecular masses identifies the genus, species, subspecies, serotype or genotype of said one or more bioagents.