Methods for concurrent identification and quantification of an unknown bioagent

• US 8,187,814 B2
• Filed: 01/08/2010
• Issued: 05/29/2012
• Est. Priority Date: 02/18/2004
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Sampath, Rangarajan, Blyn, Lawrence B., Hofstadler, Steven A., Hall, Thomas A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/684,742
• Publication Number:
  US 20120107795A1
• Time in Patent Office: 872 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

The present invention provides methods for the quantification of an unknown bioagent in a sample by amplification of nucleic acid of the bioagent, and concurrent amplification of a known quantity of a calibration polynucleotide from which are obtained a bioagent identifying amplicon and a calibration amplicon. Upon molecular mass analysis, mass and abundance data are obtained. The identity of the bioagent is then determined from the molecular mass of the bioagent identifying amplicon and the quantity of the identified bioagent in the sample is determined from the abundance data of the bioagent identifying amplicon and the abundance data of the calibration amplicon.

Claims

1. A method for simultaneous analysis of the identity and quantity of a bioagent in a sample comprising:

contacting nucleic acid of said sample with;

a pair of primers designed to produce a bioagent identifying amplicon from said nucleic acid under amplification conditions; and

a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of amplification with said primers under said amplification conditions;

concurrently amplifying said nucleic acid and said calibration sequence with said pair of primers in the same amplification mixture to obtain a first amplification product comprising a bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;

obtaining molecular mass data and abundance data for said first and second amplification products in said amplification mixture wherein the 5′ and

3′

ends of said amplification products are the sequences of said pair of primers or complements thereof;

distinguishing said first and second amplification products based on their respective molecular masses;

comparing the molecular mass of said first amplification product with a database of molecular masses of bioagent identifying amplicons for known bioagents wherein a match between the molecular mass of said first amplification product and a molecular mass of a bioagent identifying amplicon for a known bioagent in a database indicates the identity of said bioagent, and a molecular mass of said second amplification product identifies said calibration amplicon; and

calculating the quantity of said bioagent from said abundance data of said first and second amplification products.

2. A method for determining the identity and quantity of a bioagent in a sample comprising:

contacting said sample with a pair of primers and a known quantity of a calibration polynucleotide comprising a calibration sequence;

concurrently amplifying nucleic acid from said bioagent in said sample with said pair of primers and amplifying nucleic acid from said calibration polynucleotide in said sample with said pair of primers to obtain a first amplification product comprising a bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;

obtaining molecular mass and abundance data for said bioagent identifying amplicon and for said calibration amplicon wherein the 5′ and

3′

ends of said bioagent identifying amplicon and said calibration amplicon are the sequences of said pair of primers or complements thereof; and

distinguishing said bioagent identifying amplicon from said calibration amplicon based on their respective molecular masses; and

comparing a molecular mass of said bioagent identifying amplicon with a database of molecular masses of bioagent identifying amplicons for known bioagents wherein a match between a molecular mass of said first amplification product and a molecular mass of a bioagent identifying amplicon for a known bioagent in a database indicates the identity of said bioagent, and a molecular mass of said second amplification product identifies said calibration amplicon, and wherein comparison of bioagent identifying amplicon abundance data and calibration amplicon abundance data indicates the quantity of bioagent in said sample.

3. The method of claim 2 wherein said bioagent is a bacterium or a virus.

4. The method of claim 2 wherein said calibration sequence comprises a chosen standard sequence of a bioagent identifying amplicon with the exception of a deletion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

5. The method of claim 2 wherein said calibration sequence comprises a chosen standard sequence of a bioagent identifying amplicon with the exception of an insertion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

6. The method of claim 2 wherein said calibration sequence has at least 80% sequence identity with a chosen standard sequence of a bioagent identifying amplicon.

7. The method of claim 2 wherein said calibration polynucleotide is present within a vector.

8. The method of claim 2 wherein said molecular mass is obtained by mass spectrometry.

9. The method of claim 2 wherein said molecular mass is obtained by ESI-FTICR or ESI-TOF mass spectrometry.

10. The method of claim 2 comprising construction of a standard curve wherein the amount of said calibration polynucleotide in said sample is varied.

11. The method of claim 2 comprising multiplex amplification wherein a plurality bioagent identifying amplicons are amplified with a plurality of primer pairs which amplify corresponding calibration sequences.

12. The method of claim 11 wherein said plurality of primer pairs comprise survey primers, division-wide primers, clade group primers and sub-species characteristic primers.

13. The method of claim 2 comprising amplification of a plurality of bioagent identifying amplicons within a plurality of core genes with a plurality of primer pairs.

14. The method of claim 13 wherein said primer pairs hybridize to conserved regions of nucleic acid of genes encoding 16S and 23S rRNAs, RNA polymerase subunits, t-RNA synthetases, elongation factors, ribosomal proteins, protein chain initiation factors, cell division proteins, chaperonin groEL, chaperonin dnaK, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, and DNA topoisomerases.

15. A method for simultaneous analysis of the identity and quantity of a bioagent in a sample comprising:

contacting nucleic acid of said sample with;

a pair of primers designed to produce a bioagent identifying amplicon from said nucleic acid under amplification conditions; and

a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of amplification with said primers under said amplification conditions;

concurrently amplifying said nucleic acid and said calibration sequence with said pair of primers in the same amplification mixture to obtain a first amplification product comprising a bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;

obtaining molecular mass data and abundance data for said first and second amplification products in said amplification mixture wherein the 5′ and

3′

ends of said amplification products are the sequences of said pair of primers or complements thereof;

determining the base compositions of said first and second amplification products based on their respective molecular masses;

comparing a base composition of said first amplification product with a database of base compositions of bioagent identifying amplicons for known bioagents wherein a match between a base composition of an amplification product and a base composition of a bioagent identifying amplicon for a known bioagent in a database of base compositions indicates the identity of said bioagent, and a base composition of said second amplification product identifies said calibration amplicon; and

calculating the quantity of said bioagent from said abundance data of said first and second amplification products.

16. A method for determining the identity and quantity of a bioagent in a sample comprising:

contacting said sample with a pair of primers and a known quantity of a calibration polynucleotide comprising a calibration sequence;

concurrently amplifying nucleic acid from said bioagent in said sample with said pair of primers and amplifying nucleic acid from said calibration polynucleotide in said sample with said pair of primers to obtain a first amplification product comprising a bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;

obtaining molecular mass and abundance data for said bioagent identifying amplicon and for said calibration amplicon wherein the 5′ and

3′

ends of said bioagent identifying amplicon and said calibration amplicon are the sequences of said pair of primers or complements thereof;

determining the base compositions of said bioagent identifying amplicon and said calibration amplicon based on their respective molecular masses;

comparing a base composition of said bioagent identifying amplicon with a database of base compositions of bioagent identifying amplicons for known bioagents wherein a match between a base composition of said bioagent identifying amplicon and a base composition of a bioagent identifying amplicon for a known bioagent in a database of base compositions indicates the identity of said bioagent, and a base composition of said second amplification product identifies said calibration amplicon, and wherein comparison of bioagent identifying amplicon abundance data and calibration amplicon abundance data indicates the quantity of bioagent in said sample.

17. The method of claim 16 wherein said bioagent is a bacterium or a virus.

18. The method of claim 16 wherein said calibration sequence comprises a chosen standard sequence of a bioagent identifying amplicon with the exception of a deletion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

19. The method of claim 16 wherein said calibration sequence comprises a chosen standard sequence of a bioagent identifying amplicon with the exception of an insertion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

20. The method of claim 16 wherein said calibration sequence has at least 80% sequence identity with a chosen standard sequence of a bioagent identifying amplicon.

21. The method of claim 16 wherein said calibration polynucleotide is present within a vector.

22. The method of claim 16 wherein said molecular mass is obtained by mass spectrometry.

23. The method of claim 16 wherein said molecular mass is obtained by ESI-FTICR or ESI-TOF mass spectrometry.

24. The method of claim 16 comprising construction of a standard curve wherein the amount of said calibration polynucleotide in said sample is varied.

25. The method of claim 16 comprising multiplex amplification wherein a plurality of bioagent identifying amplicons are amplified with a plurality of primer pairs which amplify corresponding calibration sequences.

26. The method of claim 25 wherein said plurality of primer pairs comprise survey primers, division-wide primers, clade group primers and sub-species characteristic primers.

27. The method of claim 16 comprising amplification of a plurality of bioagent identifying amplicons within a plurality of core genes with a plurality of primer pairs.

28. The method of claim 27 wherein said primer pairs hybridize to conserved regions of nucleic acid of genes encoding 16S and 23S rRNAs, RNA polymerase subunits, t-RNA synthetases, elongation factors, ribosomal proteins, protein chain initiation factors, cell division proteins, chaperonin groEL, chaperonin dnaK, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, and DNA topoisomerases.

29. The method of claim 16 wherein said base compositions are determined from said molecular masses by base composition probability cloud analysis.

30. The method of claim 25 wherein said plurality of primer pairs are selected by base composition cloud analysis.