COMPOSITIONS AND METHOD FOR RAPID, REAL-TIME DETECTION OF INFLUENZA A VIRUS (H1N1) SWINE 2009

• US 20100009343A1
• Filed: 07/28/2009
• Published: 01/14/2010
• Est. Priority Date: 09/12/2006
• Status: Active Grant

• Current Assignee: Longhorn Vaccines and Diagnostics, LLC
• Original Assignee: Longhorn Vaccines and Diagnostics, LLC
• Inventors: Fischer, Gerald W., Daum, Luke T.
• Granted Patent: US 8,097,419 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

Disclosed are oligonucleotide amplification primers and detection probes specific for the amplification and detection of pathogenic organisms, including for example, specific Influenza A H1N1 viral isolates. Also disclosed is a biological organism identification kit including the disclosed nucleic acid probes and primers, as well as thermal cycling reagents that is both portable and durable, and may also be self-contained for remote, or in-field analysis and identification of particular influenza isolates from a variety of biological specimen types.

Claims

1. A method for detecting the presence or absence of an Influenza A H1N1 subtype virus-specific nucleic acid segment in a population of polynucleotides in a sample, which method comprises:

(a) performing at least one cycling step, wherein the cycling comprises at least a first amplifying step and at least a first hybridizing step, wherein the at least a first amplifying step comprises contacting the sample with a pair of different, independently selected Influenza A H1N1 subtype virus-specific amplification primers to produce an Influenza A H1N1 subtype virus-specific amplification product if an Influenza A H1N1 subtype virus-specific nucleic acid segment is present in the sample; and

(b) detecting the presence of the amplification product using a labeled oligonucleotide probe that is specific for the H1N1 amplification product, wherein the presence of the amplification product is indicative of the presence of one or more Influenza A H1N1-specific nucleic acid segments in the population of polynucleotides.

2. The method of claim 1, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:

51).

3. The method of claim 2, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:

51).

4. The method of claim 3, wherein the pair of amplification primers comprises a first oligonucleotide primer that consists essentially of the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:

51).

5. The method of claim 1, wherein the pair of amplification primers comprises a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence AATCCTGTRGCCAGTCTCAATTTTG (SEQ ID NO:

52).

6. The method of claim 5, wherein the pair of amplification primers comprises a second oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence AATCCTGTRGCCAGTCTCAATTTTG (SEQ ID NO:

52).

7. The method of claim 1, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:

51, and a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

52.

8. The method of claim 7, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:

51, and a second oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

52.

9. The method of claim 1, wherein the detection probe comprises a first oligonucleotide probe of less than about 50 nucleotides in length, and further wherein the first oligonucleotide probe comprises the nucleic acid sequence of TCCAAAATATGTAAAAAG (SEQ ID NO:

53).

10. The method of claim 9, wherein the detection probe comprises a first oligonucleotide probe of less than about 30 nucleotides in length, and further wherein the first oligonucleotide probe comprises the nucleic acid sequence TCCAAAATATGTAAAAAG (SEQ ID NO:

53).

11. The method of claim 1, wherein (a) the pair of amplification primers comprises:

(i) a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

51, and (ii) a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

52; and

(b) the detection probe comprises a first oligonucleotide probe of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

53.

12. The method of claim 11, wherein (a) the pair of amplification primers comprises:

(i) a first oligonucleotide primer that consists essentially of the nucleic acid sequence of SEQ ID NO;

51, and (ii) a second oligonucleotide primer that consists essentially of the nucleic acid sequence of SEQ ID NO;

52; and

(b) the detection probe comprises a first oligonucleotide probe that consists essentially of the nucleic acid sequence of SEQ ID NO;

53.

13. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, cells, tissues, and serum.

14. An Influenza A H1N1 Virus-specific oligonucleotide amplification primer set, wherein the first amplification primer is less than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO:

51 and the second amplification primer is less than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO;

52.

15. The Influenza A H1N1 Virus-specific oligonucleotide amplification set of claim 14, further comprising a first detection probe, wherein the first detection probe comprises a labeled oligonucleotide probe of less than about 50 nucleotides in length that comprises the nucleotide sequence of SEQ ID NO:

53.

16. A kit comprising, in suitable container means, the Influenza A H1N1 Virus-specific oligonucleotide amplification primer set of claim 14, and instructions for using the primer set in a PCR amplification of a population of polynucleotides obtained from a biological sample or specimen.

17. The kit of claim 16, further comprising, in suitable container means, an oligonucleotide detection probe that specifically binds to the amplification product produced from PCR amplification of a population of polynucleotides obtained from a biological sample or specimen that contains, or is suspected of containing, an Influenza A H1N1 Virus-specific nucleic acid segment.

18. An article of manufacture, comprising:

a pair of Influenza A H1N1 Virus-specific oligonucleotide amplification primers; and

a first Influenza A H1N1 Virus-specific oligonucleotide detection probe;

wherein the detection probe comprises at least one detectable label.

19. The article of manufacture of claim 18, further comprising a package insert having instructions for using the pair of primers and the detection probe to detect the presence or absence of an Influenza A H1N1 Virus-specific nucleic acid segment within a population of polynucleotides obtained from a biological sample that was collected from a human subject.

20. A composition comprising:

(a) a first pair of Influenza A H1N1 Virus-specific amplification primers, wherein the pair of primers comprises;

(i) a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

51; and

(ii) a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO;

52; and

(b) a first Influenza A H1N1 Virus-specific oligonucleotide detection probe, comprising;

(i) a first oligonucleotide detection probe of less than about 50 nucleotides in length, wherein the probe comprises the nucleic acid sequence of SEQ ID NO;

53; and

(ii) at least a first detection reagent operably linked to the oligonucleotide detection probe.

21. The composition of claim 20, wherein the polynucleotide is amplified from a population of polynucleotides obtained from a biological sample.

22. The composition of claim 21, wherein the polynucleotide is amplified from a population of polynucleotides obtained from a human suspected of infection by an H1N1 subtype of Influenza A Virus.

23. The composition of claim 20, wherein the oligonucleotide detection probe comprises a radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin-resonance label.

24. The composition of claim 20, further comprising one or more components, buffers, enzymes, or reagents for performing polymerase chain reaction.

25. The composition of claim 20, wherein the composition is at least substantially stable at room temperature and is adapted and configured for use with a polymerase chain reaction (PCR) device.

27. A method of identifying a subtype of Influenza A virus comprising:

detecting the presence of an Influenza A virus-specific nucleic acid segment, if present, in a population of polynucleotides using a labeled oligonucleotide probe that is specific for the Influenza A virus-specific nucleic acid segment; and

if an Influenza A virus-specific nucleic acid segment is present in the population of polynucleotides, then further identifying particular subtype of Influenza A virus using a labeled oligonucleotide probe that is specific for an H5 or an H1N1 subtype of Influenza A virus.

26. A biological organism identification product that comprises:

(a) a collection device to collect one or more sample organisms;

(b) a fixing and transporting composition present in an amount sufficient to kill the one or more sample organisms associated with the collection device;

(c) an extraction member to extract a sufficient amount of genomic nucleic acid from the one or more sample organisms to facilitate identification thereof; and

(d) a substantially stable polymerase chain reaction (PCR) component into which the sufficient amount of genomic nucleic acid can be added, and further wherein the PCR component comprises the composition of claim 30.

28. The method of claim 27, wherein the further identifying is performed by contacting the nucleic acid segment with a labeled oligonucleotide probe that is specific for an Influenza A H5 subtype, or an Influenza A H1N1 (Swine) subtype, and detecting the presence of the labeled hybridization product so produced.

29. The method of claim 28, wherein the detection of the Influenza A H5 subtype and the Influenza A H1N1 (Swine) subtype is sequential.

30. The method of claim 29, wherein the labeled oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of 5′

-TCCAAAATATGTAAAAAG-3′

(SEQ ID NO;

53), 5′

-TCAGGCCCCCTCAAAGC-3′

(SEQ ID NO;

54);

5′

-ATGGGAAATTCAGCTCT-3′

(SEQ ID NO;

55);

5′

-TCTCCAAAGTATGTCAGG-3′

(SEQ ID NO;

56);

5′

-TGAGATCAGATGCACCCAT-3′

(SEQ ID NO;

57);

5′

-AGAGRGGAAATAAGTGG-3′

(SEQ ID NO;

58), and any combination thereof.

31. The method of claim 30, wherein the oligonucleotide probe is labeled at its 5′

-end with FAM.