Respiratory infection assay

• US 10,294,534 B2
• Filed: 12/10/2012
• Issued: 05/21/2019
• Est. Priority Date: 12/09/2011
• Status: Active Grant

• Current Assignee: Secretary of State for Health and Social Care
• Original Assignee: Secretary of State for Health
• Inventors: Curran, Martin
• Primary Examiner(s): Kapushoc, Stephen T
• Application Number:
  US14/363,533
• Publication Number:
  US 20150099261A1
• Time in Patent Office: 2,353 Days
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

The present invention provides nucleic acid products and corresponding methods for screening a biological sample for the presence of a respiratory infection-causing microorganism.

Claims

1. A quantitative uniplex method for detecting the presence of one or more of at least six respiratory infection-causing microorganisms in a sample or detecting the absence of said respiratory infection-causing microorganisms in said sample, said method comprising:

A) applying a sample to a test card, wherein said test card comprises six discrete wells;

1) a first well that includes a first probe, for detecting the presence or absence of Respiratory syncytial virus (RSV A &

B), wherein the first probe has a nucleic acid sequence that comprises the nucleic acid sequence TAGGCAATGCWGC (SEQ ID NO;

     124);

2) a second well that includes a second probe, for detecting the presence or absence of Rhinoviruses, wherein the second probe has a nucleic acid sequence that comprises the nucleic acid sequence TCYGGGAYGGGACCRACTA (SEQ ID NO;

     125);

3) a third well that includes a third probe, for detecting the presence or absence of Human Metapneumovirus (hMPV), wherein the third probe has a nucleic acid sequence that comprises the nucleic acid sequence TCAGCACCAGACACACC (SEQ ID NO;

     126);

4) a fourth well that includes a fourth probe, for detecting the presence or absence of Influenza B (Flu B Quad), wherein the fourth probe has a nucleic acid sequence that comprises the nucleic acid sequence TCTGGTCATTGGRGCC (SEQ ID NO;

     127);

5) a fifth well that includes a fifth probe, for detecting the presence or absence of Influenza A (Flu A CDC DC), wherein the fifth probe has a nucleic acid sequence that comprises the nucleic acid sequence CGCTCACTGGGCACGGT (SEQ ID NO;

     128);

6) a sixth well that includes a sixth probe, for detecting the presence or absence of Influenza A subtype H5 (H5 FRET), wherein the sixth probe has a nucleic acid sequence that comprises the nucleic acid sequence AACTGRGTGTTCATTTTGT (SEQ ID NO;

     129);

B) allowing nucleic acid present in the sample to contact with the probes within said wells;

C) detecting for the presence of bound nucleic acid complex in which sample nucleic acid has bound to one or more of said probes;

wherein the presence of bound nucleic acid complex confirms that nucleic acid from one or more of said six respiratory infection-causing microorganisms is present within the sample, and wherein the absence of bound nucleic acid complex confirms that nucleic acid from all of said six respiratory infection-causing microorganisms is absent within the sample and wherein the probes comprise a detectable label and/or a detectable tag.

2. The quantitative method according to claim 1, wherein the test card further comprises a seventh well that includes a seventh probe, for detecting the presence or absence of Human parainfluenza virus type 1 (HPIV 1), wherein the seventh probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

7.

3. The method according to claim 1, wherein the test card comprises each of an additional seventh through to thirteenth wells and a corresponding seventh through to thirteenth additional probes.

4. The quantitative method according to claim 3, wherein the test card comprises one or more additional wells and corresponding one or more additional probes selected from:

14) a fourteenth well that includes a fourteenth probe, for detecting the presence or absence of Respiratory syncytial virus (RSV #2);

(RSV #3), wherein the fourteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

14;

15) a fifteenth well that includes a fifteenth probe, for detecting the presence or absence of Enteroviruses, wherein the fifteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

15;

16) a sixteenth well that includes a sixteenth probe, for detecting the presence or absence of Severe Acute Respiratory Syndrome coronavirus (SARS), wherein the sixteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

16;

17) a seventeenth well that includes a seventeenth probe, for detecting the presence or absence of Group 2 Coronaviruses OC43 and HKU1 (GP2 OC43/HKU1), wherein the seventeenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

17;

18) an eighteenth well that includes an eighteenth probe, for detecting the presence or absence of Group 1 Coronavirus NL63 (GP1 NL63), wherein the eighteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

18;

19) a nineteenth well that includes a nineteenth probe, for detecting the presence or absence of Group 1 Coronavirus 229E (GP1 229E), wherein the nineteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

19;

20) a twentieth well that includes a twentieth probe, for detecting the presence or absence of Human Adenoviruses, wherein the twentieth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

20;

21) a twenty first well that includes a twenty first probe, for detecting the presence or absence of Bocavirus, wherein the twenty first probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

21;

22) a twenty second well that includes a twenty second probe, for detecting the presence or absence of Influenza A (Flu A Quad), wherein the twenty second probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

22;

23) a twenty third well that includes a twenty third probe, for detecting the presence or absence of Influenza A H1 2009 (H1 sw

     2009), wherein the twenty third probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

23;

24) a twenty fourth well that includes a twenty fourth probe, for detecting the presence or absence of Influenza A N1 2009 (N1 CFI), wherein the twenty fourth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

24;

25) a twenty fifth well that includes a twenty fifth probe, for detecting the presence or absence of Influenza A H1 Seasonal (H1 seasonal CFI), wherein the twenty fifth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

25;

26) a twenty sixth well that includes a twenty sixth probe, for detecting the presence or absence of Influenza A H3 Seasonal (H3 seasonal CFI), wherein the twenty sixth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

26;

27) a twenty seventh well that includes a twenty seventh probe, for detecting the presence or absence of Influenza A H5 (H5 CFI), wherein the twenty seventh probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

27;

28) a twenty eighth well that includes a twenty eighth probe, for detecting the presence or absence of Influenza A H7 (H7), wherein the twenty eighth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

28;

29) a twenty ninth well that includes a twenty ninth probe, for detecting the presence or absence of Influenza A H9 (H9 a), wherein the twenty ninth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

29;

30) a thirtieth well that includes a thirtieth probe, for detecting the presence or absence of Influenza A H9 (H9 b), wherein the thirtieth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

30;

31) a thirty first well that includes a thirty first probe, for detecting the presence or absence of Human parainfluenza virus type 1 (HPIV 1 #2), wherein the thirty first probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

31;

32) a thirty second well that includes a thirty second probe, for detecting the presence or absence of Human parainfluenza virus type 3 (HPIV 1 #3), wherein the thirty second probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

32;

33) a thirty third well that includes a thirty third probe, for detecting the presence or absence of Rhinoviruses #2, wherein the thirty third probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

33.

5. The method according to claim 4, wherein the test card comprises each of the additional fourteenth through to thirty third wells and the corresponding fourteenth through to thirty third additional probes.

6. The quantitative method according to claim 5, wherein the test card comprises one or more additional wells and corresponding one or more additional probes selected from:

34) a thirty fourth well that includes a thirty fourth probe, for detecting the presence or absence of Legionella pneumophilia, wherein the thirty fourth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

34;

35) a thirty fifth well that includes a thirty fifth probe, for detecting the presence or absence of Mycoplasma pneumoniae, wherein the thirty fifth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

35;

36) a thirty sixth well that includes a thirty sixth probe, for detecting the presence or absence of Chlamydiophilia pneumoniae, wherein the thirty sixth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

36;

37) a thirty seventh well that includes a thirty seventh probe, for detecting the presence or absence of Coxiella burnetti, wherein the thirty seventh probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

37;

38) a thirty eighth well that includes a thirty eighth probe, for detecting the presence or absence of Chlamydiophilia psittaci, wherein the thirty eighth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

38;

39) a thirty ninth well that includes a thirty ninth probe, for detecting the presence or absence of Chlamydiophilia abortus, wherein the thirty ninth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

     39).

7. The method according to claim 6, wherein the test card comprises each of the additional thirty third through to thirty ninth wells and the corresponding thirty third through to thirty ninth additional probes.

8. The method according to claim 7, wherein the test card comprises one or more additional control wells and corresponding one or more additional control probes selected from:

1) a fortieth well that includes a fortieth probe, for detecting the presence or absence of Escherichia coli Bacteriophage MS2 (MS2 IC), wherein the fortieth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

40;

2) a forty first well that includes a forty first probe, for detecting the presence or absence of Human Ribonuclease P gene (RNAse P), wherein the forty first probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

41.

9. The method according to claim 8, wherein the test card comprises one or both of the additional fortieth or forty first well and the corresponding fortieth through forty first additional probes.

10. The method according to claim 1, wherein said method includes a nucleic acid amplification step prior to detection.

11. The quantitative method according to claim 2, wherein the test card further comprises:

an eighth well that includes an eighth probe, for detecting the presence or absence of Human parainfluenza virus type 2 (HPIV

     2),wherein the eighth probe has a nucleic acid sequence that comprises the nucleic acid sequence of SEQ ID NO;

131.

12. The quantitative method according to claim 11, wherein the test card further comprises:

a ninth well that includes a ninth probe, for detecting the presence or absence of Human parainfluenza virus type 3 (HPIV

     3), wherein the ninth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

9.

13. The quantitative method according to claim 12, wherein the test card further comprises:

a tenth well that includes a tenth probe, for detecting the presence or absence of Human parainfluenza virus type 4 (HPIV

     4), wherein the tenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

10.

14. The quantitative method according to claim 13, wherein the test card further comprises:

eleventh well that includes an eleventh probe, for detecting the presence or absence of Human Adenoviruses #2, wherein the eleventh probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

11.

15. The quantitative method according to claim 14, wherein the test card further comprises:

a twelfth well that includes a twelfth probe, for detecting the presence or absence of Tamiflu sensitive Influenza A H1 2009 wherein the twelfth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

12.

16. The quantitative method according to claim 15, wherein the test card further comprises:

a thirteenth well that includes a thirteenth probe, for detecting the presence or absence of Tamiflu resistant Influenza A H1 2009, wherein the thirteenth probe has a nucleic acid sequence that comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO;

13.

17. The quantitative method according to claim 1, wherein the detectable label and/or the detectable tag comprises a radiolabel, a fluorescent molecule, a coloured molecule, an enzymatic marker or a chromogenic marker.

18. The quantitative method according to claim 1, wherein the detectable label is selected from a group consisting of Fam, a minor groove binder (MGB), digoxygenin, fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa 532, and Cy3.