Rapid, informative diagnostic assay for influenza viruses including H5N1

• US 7,989,185 B2
• Filed: 11/27/2007
• Issued: 08/02/2011
• Est. Priority Date: 11/29/2006
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Kumm, Jochen, Diamond, Lisa, Davis, Ronald W., Pourmand, Nader
• Primary Examiner(s): Kim; Young J
• Application Number:
  US11/945,960
• Publication Number:
  US 20090123909A1
• Time in Patent Office: 1,344 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

A rapid diagnostic assay for influenza virus, particularly avian influenza and more particularly H5N1, is described. The assay is based on amplification of a significant portion of the hemagglutinin (HA) gene and sequencing of several loci within the HA gene, using techniques which can obtain real time sequence information from multiple sites of a target DNA, in particular pyrosequencing and bioluminescence regenerative cycle. The assay contemplates the use of information-rich subsequences within the HA gene, e.g., (1) a glycosylation sequon; (2) receptor binding site; and (3) HA1/HA2 cleavage site. Other subsequences for sequencing include strain and clade markers, which vary among H5N1 strains.

Claims

1. A method for detecting the presence of an influenza A virus and identifying its strain by determining selected subsequences of its hemagglutinin (HA) gene, comprising:

(a) obtaining a sample possibly containing an influenza A virus;

(b) obtaining DNA having a sequence from an HA gene in a virus in the sample;

(c) obtaining sequence information for at least three portions of the DNA from step (b), wherein said three portions are (i) a glycosylation sequon;

(ii) a receptor-binding site; and

(iii) an HA1/HA2 cleavage site, whereby said sequence information from said three portions together detects the strain of the influenza A virus.

2. The method of claim 1 wherein said sequence information is obtained by sequencing less than 30 bases for each of said (i) a glycosylation sequon;

(ii) receptor-binding site; and

(iii) HA1/HA2 cleavage site.

3. The method of claim 2 wherein the sequencing is done using pyrosequencing.

4. The method of claim 1 wherein obtaining sequence information further comprises a step of detecting hybridization of sample to a probe which hybridizes to a sequence of (i) a glycosylation sequon;

(ii) a receptor-binding site;

or (iii) an HA1/HA2 cleavage site.

5. The method of claim 1 wherein the DNA of step (b) is immobilized on a surface selected from a bead or a well.

6. The method of claim 1 wherein said DNA is obtained by RT-PCR.

7. The method of claim 6 wherein the RT-PCR is done with primers, which will amplify all of SEQ ID NOs 3 through 21.

8. The method of claim 1 further comprising obtaining sequence information of strain markers 1, 2 and 3, wherein strain marker 1 has a sequence selected from the group consisting of SEQ ID NO:

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38 and SEQ ID NO;

39;

strain marker 2 has a sequence selected from the group consisting of SEQ ID NO;

47, SEQ ID NO;

48, and SEQ ID NO;

49; and

strain marker 3 has a sequence selected from the group consisting of SEQ ID NO;

73, SEQ ID NO;

74, SEQ ID NO;

75, and SEQ ID NO;

76.

9. The method of claim 8 further comprising sequencing one or more regions of the HA gene indicative of clade, as set forth in Clade Marker 1 and Clade Marker 2, wherein Clade Marker 1 has a sequence selected from the group consisting of SEQ ID NO:

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31; and

Clade Marker 2 has a sequence selected from the group consisting of SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54 SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61 SEQ ID NO;

62, and SEQ ID NO;

63.

10. The method of claim 9 further comprising sequencing an HA cleavage site as identified by a sequence selected from the group consisting of SEQ ID NO:

78, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, and SEQ ID NO;

83.

11. The method of claim 1 wherein nucleotides A, T, G, and C are added in a predetermined order based on an expected sequence of regions (i) a glycosylation sequon;

(ii) a receptor-binding site; and

(iii) an HA1/HA2 cleavage site.

12. A method for analyzing an H5N1 influenza strain comprising the steps of obtaining cDNA from a sample possibly containing an H5N1 influenza virus and further obtaining a cDNA sequence according to at least one marker and correlating the marker to a corresponding strain as follows:

(a) Goose/Guangdong/1/96, correlates to at least one sequence selected from the group consisting of SEQ ID NO;

25, SEQ ID NO;

32, SEQ ID NO;

40, SEQ ID NO;

47, and SEQ ID NO;

50;

(b) HongKong/156/97 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

25, SEQ ID NO;

33, SEQ ID NO;

41, SEQ ID NO;

47, SEQ ID NO;

50;

(c) Hong Kong/483/97 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

25, SEQ ID NO;

34, SEQ ID NO;

41, SEQ ID NO;

47, and SEQ ID NO;

51;

(d) HongKong/213/03 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

26, SEQ ID NO;

35, SEQ ID NO;

42 SEQ ID NO;

47, and SEQ ID NO;

51;

(e) Chicken/Korea/ES/03 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

27, SEQ ID NO;

36, SEQ ID NO;

43, SEQ ID NO;

48, and SEQ ID NO;

52,

(f) Vietnam/1203/04 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

28, SEQ ID NO;

37, SEQ ID NO;

41, SEQ ID NO;

47, and SEQ ID NO;

53,

(g) Vietnam/JP14/05 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

29, SEQ ID NO;

38, SEQ ID NO;

44, SEQ ID NO;

47, and SEQ ID NO;

54;

(h) Vietnam/HN304/08/05 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

30, SEQ ID NO;

38, SEQ ID NO;

45, SEQ ID NO;

47, and SEQ ID NO;

55; and

(i) Indonesia/5/05 correlates to at least one sequence selected from the group consisting of SEQ ID NO;

31, SEQ ID NO;

39, SEQ ID NO;

46, SEQ ID NO;

49 and SEQ ID NO;

53.