Rapid, Informative Diagnostic Assay For Influenza Viruses Including H5N1

• US 20090123909A1
• Filed: 11/27/2007
• Published: 05/14/2009
• Est. Priority Date: 11/29/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Davis, Ronald W., Pourmand, Nader, Kumm, Jochen, Diamond, Lisa
• Granted Patent: US 7,989,185 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 influenza virus and determining selected subsequences of its hemagglutinin (HA) gene, comprising:

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

(b) obtaining a DNA corresponding to an HA gene in a virus in the sample;

(c) obtaining sequence information for at least three portions of the DNA from step (b) corresponding to subsequences of the HA gene comprising (i) a glycosylation sequon;

(ii) a receptor-binding site; and

(iii) an HA1/HA2 cleavage site.

2. The method of claim 1 wherein said sequence information is obtained by sequencing less than 30 bases for each of (i) (ii) and (iii).

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

4. The method of claim 1 wherein obtaining sequence information is done by detecting hybridization of sample to a probe which hybridizes to a sequence of (i) (ii) or (iii).

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 a strain markers 1, 2 and 3 as set forth in Table 1.

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 in Table 1.

10. The method of claim 9 further comprising sequencing an HA cleavage site as identified in Table 1, part B.

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

12. A kit for analyzing influenza virus, comprising:

(d) PCR primers for amplifying a substantial portion of an HA gene; and

(e) sequencing primers, binding to different strains of influenza virus, hybridizing adjacent to (1) a glycosylation sequon;

(2) a receptor-binding site; and

(3) an HA1/HA2 cleavage site.

13. The kit of claim 12 further comprising dNTPs A, T, G, and C.

14. The kit of claim 12 further comprising DNA synthesizing enzymes, an enzyme apyrase to decompose dNTP (deoxynucleotide triphosphates);

sulfurylase to convert pyrophosphate into ATP;

luciferin; and

luciferase for generating light in a sequencing reaction.

15. The kit of claim 12 wherein said sequencing primers are selected to bind at least one of a clade marker 1, strain marker 1, glycosylation sequon, strain marker 2, clade marker 2 (forward), clade marker 2 (reverse), receptor specificity site, strain marker 3, or cleavage site each as identified in Table 1.

16. The kit of claim 12 further comprising at least one sequencing primer having a sequence at least 90% identical to a sequence listed as an internal primer in FIG. 5.

17. A method for analyzing an H5N1 influenza strain comprising the step of obtaining a cDNA sequence according to at least one marker listed in Table 1 and correlating the marker to a corresponding strain as listed in that Table.

18. A method for analyzing an H5N1 influenza strain comprising the step of obtaining an amino acid sequence from the strain and comparing it to at least three of four amino acid sequences for HA clade, glycosylation sequon, receptor binding site and cleavage motif, as set forth in Table 1.

19. PCR primers for amplifying an influenza virus HA gene comprising SEQ ID NO:

22 and SEQ ID NO;

23.

20. The PCR primers of claim 19 further comprising an internal primer to a subsequence found in SEQ ID) NOs:

13, 14 and 15.