Methods for concurrent identification and quantification of an unknown bioagent

• US 7,666,592 B2
• Filed: 02/17/2005
• Issued: 02/23/2010
• Est. Priority Date: 02/18/2004
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hofstadler, Steven A., Sampath, Rangarajan, Hall, Thomas A., Blyn, Lawrence B., Ecker, David J.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US11/059,776
• Publication Number:
  US 20090004643A1
• Time in Patent Office: 1,832 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 polymerase chain reaction amplification conditions; and

a known quantity of a calibration polynucleotide comprising a calibration sequence designed to produce a calibration amplicon as a result of polymerase chain reaction 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 polymerase chain reaction amplification product comprising a bioagent identifying amplicon and a second polymerase chain reaction amplification product comprising a calibration amplicon;

obtaining molecular mass data and abundance data for said first and second polymerase chain reaction 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 polymerase chain reaction amplification products based on their respective molecular masses, wherein the molecular mass of said first polymerase chain reaction amplification product identifies said bioagent, and the molecular mass of said second polymerase chain reaction amplification product identifies said calibration amplicon; and

calculating the quantity of said bioagent from said abundance data of said first and second polymerase chain reaction 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 a polymerase chain reaction and said pair of primers and amplifying nucleic acid from said calibration polynucleotide in said sample with a polymerase chain reaction and said pair of primers to obtain a first polymerase chain reaction amplification product comprising a bioagent identifying amplicon and a second polymerase chain reaction 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 amplicons 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, wherein the molecular mass of said bioagent identifying amplicon indicates the identity of said bioagent, and 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 wherein said molecular mass of said bioagent identifying amplicon identifies said bioagent as a result of determination of the base composition of said bioagent identifying amplicon.

11. A method for determining the identity and quantity of a bacterium or a virus 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 bacterium or virus in said sample with a polymerase chain reaction and said pair of primers and amplifying nucleic acid from said calibration polynucleotide in said sample with a polymerase chain reaction and said pair of primers to obtain a first polymerase chain reaction amplification product comprising a bacterial or viral bioagent identifying amplicon and a second polymerase chain reaction amplification product comprising a calibration amplicon;

obtaining the molecular mass and abundance data for said bacterial or viral 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 bacterial or viral bioagent identifying amplicon from said calibration amplicon based on their respective molecular masses, wherein the molecular mass of said bacterial or viral bioagent identifying amplicon indicates the identity of said bacterium or virus, and comparison of bacterial or viral bioagent identifying amplicon abundance data and calibration amplicon abundance data indicates the quantity of bacterium or virus in said sample.

12. The method of claim 11 wherein said virus is an RNA virus whose RNA is reverse transcribed to DNA which represents said nucleic acid amplified by said pair of primers.

13. The method of claim 12 wherein said reverse transcribing is accomplished using reverse transcriptase.

14. The method of claim 11 wherein said virus is a DNA virus whose DNA represents said nucleic acid amplified by said primers.

15. The method of claim 11 wherein said calibration sequence comprises the sequence of a chosen standard sequence of a bacterial or viral bioagent identifying amplicon with the exception of a deletion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

16. The method of claim 11 wherein said calibration sequence comprises the sequence of a chosen standard sequence of a bacterial or viral bioagent identifying amplicon with the exception of an insertion of about 2 to about 8 consecutive nucleotide residues of said standard sequence.

17. The method of claim 11 wherein said calibration sequence has at least 80% sequence identity with a chosen standard sequence of a viral bioagent identifying amplicon.

18. The method of claim 11 wherein said calibration polynucleotide is present within a vector.

19. The method of claim 11 wherein said molecular mass is obtained by mass spectrometry.

20. The method of claim 11 wherein said molecular mass is obtained by ESI-FTICR or ESI-TOF mass spectrometry.

21. 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, wherein the molecular mass of said first amplification product identifies said bioagent, and the 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.

22. A method for determining the identity and quantity of a bioagent in a sample consisting of:

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, wherein the molecular mass of said bioagent identifying amplicon indicates the identity of said bioagent, and comparison of bioagent identifying amplicon abundance data and calibration amplicon abundance data indicates the quantity of bioagent in said sample.

23. The method of claim 22 wherein said bioagent is a bacterium or a virus.

24. The method of claim 22 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.

25. The method of claim 22 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.

26. The method of claim 22 wherein said calibration sequence has at least 80% sequence identity with a chosen standard sequence of a bioagent identifying amplicon.

27. The method of claim 22 wherein said calibration polynucleotide is present within a vector.

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

29. The method of claim 22 wherein said molecular mass is obtained by ESI-FTICR or ESI-TOF mass spectrometry.

30. The method of claim 22 wherein said molecular mass of said bioagent identifying amplicon identifies said bioagent as a result of determination of the base composition of said bioagent identifying amplicon.