METHODS AND COMPOSITIONS FOR SEQUENCE-SPECIFIC PURIFICATION AND MULTIPLEX ANALYSIS OF NUCLEIC ACIDS

• US 20120045756A1
• Filed: 05/18/2011
• Published: 02/23/2012
• Est. Priority Date: 05/19/2010
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: NAZARENKO, Irina, VIRMANI, Arvind, ROTHMANN, Thomas, O'NEIL, Dominic, LOWE, Brian, AGARWAL, Shiuli, BASHAM, Holly
• Granted Patent: US 9,422,593 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods and materials for determining the presence of at least one nucleic acid in a sample are provided, said methods comprising (1) a purification step using sequence specific hybrid capture; (2) an amplification step; and (3) a detection step comprising contacting the target nucleic acid with a plurality of detectably labeled nucleic acid detection probes, wherein each (a) bears a different detectable label from the other detection probes, and/or (b) has a different melting temperature from probes bearing the same detectable label. Also disclosed are compositions and kits for use in such a method.

Claims

1. A method of detecting at least one target nucleic acid in a sample, said method comprising:

A. purifying the at least one target nucleic acid by;

A1. generating a double-stranded nucleic acid hybrid of the at least one target nucleic acid by hybridizing the at least one target nucleic acid to a hybrid probe set comprising at least a first nucleic acid probe specific for the at least one target nucleic acid;

A2. separating the double-stranded nucleic acid hybrid from the sample to generate at least one purified nucleic acid;

B. amplifying at least a portion of the at least one purified nucleic acid to generate an amplified nucleic acid; and

C. detecting the target nucleic acid by;

C1. contacting the amplified nucleic acid with at least one detection probe set, wherein;

C1(a). each detection probe of the detection probe set bears a detectable label;

C1(b). at least two detection probes of the detection probe set carry the same detectable label; and

C1(c). each probe carrying the same detectable label has a melting temperature (T_m) which differs from the other probes with the same label;

C2. detecting and identifying the detectable label of a detection probe hybridized to the amplified nucleic acid; and

C3. detecting the temperature at which the detection probe dissociates from the amplified nucleic acid;

wherein identity of the target nucleic acid may be determined from the identification of the detectable label and the temperature at which the detection probe dissociates from the amplified nucleic acid.

2. The method of claim 1, wherein the double-stranded nucleic acid hybrid is separated from the sample by contacting the double stranded nucleic acid hybrid with a hybrid specific molecule.

3. The method of claim 2, wherein binding of the hybrid specific molecule immobilizes the target nucleic acid to a solid phase.

4. The method of claim 3, wherein the hybrid specific molecule is bound to or adapted to be bound to a solid support.

5. The method of claim 3, wherein the target nucleic acid is amplified while immobilized to the solid phase.

6. The method of claim 2, wherein the double stranded nucleic acid hybrid is a DNA:

RNA hybrid and the hybrid specific molecule is an anti-DNA;

RNA hybrid antibody or a fragment thereof.

7. The method of claim 1, wherein the target nucleic acid is amplified by an isothermal amplification.

8. The method of claim 7, wherein the isothermal amplification utilizes:

(a) a polymerase having strand displacement activity; and

(b) random primers.

9. The method of claim 8, wherein the isothermal amplification comprises whole genome amplification.

10. The method of claim 1, wherein each probe comprising the same detectable label differs with respect to their melting temperature by at least 0.1°

C.

11. The method of claim 1, wherein each probe comprising the same detectable label differs with respect to their melting temperature from 0.5°

C. to 10°

C.

12. The method of claim 1, wherein each probe comprising the same detectable label differs with respect to their melting temperature by about 5°

C.

13. The method of claim 1, wherein each probe of the detection probe set comprises at least one fluorophore selected from the group consisting of:

c1(d);

a fluorophore capable of being excited by an excitation source of 365±

20 nm and detected using a 460±

15 nm detection filter;

c1(e);

a fluorophore capable of being excited by an excitation source of 470±

10 nm and detected using a 510±

5 nm detection filter;

c1(f);

a fluorophore capable of being excited by an excitation source of 530±

5 nm and detected using a 555±

5 nm detection filter;

c1(g);

a fluorophore capable of being excited by an excitation source of 585±

5 nm and detected using a 610±

5 nm detection filter;

c1(h);

a fluorophore capable of being excited by an excitation source of 625±

10 nm and detected using a of 660±

10 nm detection filter; and

c1(i);

a fluorophore capable of being excited by an excitation source of 680±

5 nm and detected using a 712 nm long pass detection filter.

14. The method of claim 13, wherein the detection probe set comprises at least three groups of probes, wherein each probe of each of said at least three groups:

α

) comprises a fluorophore falling within a single group according to c1(d) to c1(i); and

β

) has a melting temperature at least 0.1°

C. different from every other probe of the same group.

15. The method of claim 1, wherein the detection probe set comprises:

c1(d);

at least one detection probes specific for a ciprofloxacin resistance target selected from the group consisting of gyrA, gyrB-1, gyrB-2, parC, and parE; and

c1(e);

at least one detection probe specific for a genomic or plasmid target selected from the group consisting of capB2, capB4, pXO1, pXO2, PLA, PIM, CAF1, tul4, and fopA.

16. The method of claim 1, wherein the detection probe set comprises at least one detection probe specific for a nucleic acid derived from a pathogen selected from the group consisting of:

Mycobacterium tuberculosis, Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B;

adenovirus, HSV1, HSV2, Influenza A/B, hMPV Parainfluenzavirus 1-4, Coronavirus, Rhinovirus, Enterovirus, Bocavirus, cytomegalovirus (CMV), HIV, H1N1, chlamydia, Neisseria gonorrhoeae, Trichomonas vaginalis, Staphylococcus aureus, SARS-associated coronavirus, Escherichia coli, Bacillus anthracis, Ebolavirus, Marburgvirus, Yersinia pestis, Vibrio cholerae, F. tularensis, Brucella, Coxiella burnetii, Machupo virus, Coccidioides immitis, Coccidioides posadasii., Burkholderia mallei, Burkholderia pseudomallei, Shigella ssp. bacterium, Rickettsia rickettsii, Rickettsia prowazekii, Chlamydophila psittaci, yellow fever virus, Japanese encephalitis virus, Rift Valley fever virus, Variola major, and Variola minor.

17. The method of claim 1, wherein the amplified nucleic acid is fragmented before detection and/or during detection.

18. The method of claim 17, wherein the amplified nucleic acid is fragmented by at least one procedure selected from the group consisting of:

A. contacting the amplified nucleic acid with a restriction enzyme or nuclease or a fragment thereof;

B. incorporating a random ribonucleotide monophosphate into the amplified nucleic acid and subjecting the resulting amplified nucleic acid to an alkali treatment; and

C. physically fragmenting the amplified nucleic acid by a procedure selected from the group consisting of sonication, nebulization, and hydrodynamic shearing.

19. The method of claim 1, wherein the amplifying and detecting are performed simultaneously.

20. The method of claim 1, wherein the temperature at which the detection probe dissociates from the amplified nucleic acid is determined by contacting the amplified nucleic acid with a double stranded nucleic acid-specific dye.

21. The method of claim 20, wherein the double stranded nucleic acid-specific dye is selected from the group consisting of SYBR®

Green I, SYBR®

Gold, ethidium bromide, propidium iodide, Pico Green, Hoechst 33258, YO-PRO-I and YO-YO-I, SYTO®

9, LC Green®

, LC Green®

Plus+, and EvaGreen™

.

22. A detection probe set comprising at least three nucleic acid probes, each comprising a detectable label, wherein:

A. at least two of the nucleic acid probes comprise the same detectable label; and

B. each probe has a melting point that differs from every other probe comprising the same detectable label.

23. The detection probe set of claim 22, wherein the melting point differs by at least 0.1°

C.

24. The detection probe set of claim 22, wherein the melting point differs by 0.5°

C. to 10°

C.

25. The detection probe set of claim 22, wherein the melting point differs by about 5°

C.

26. The detection probe set of claim 22, comprising at least two sets of probes selected from the group consisting of:

(a1);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 365±

20 nm and detected with a 460±

15 nm detection filter;

(a2);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 470±

10 nm and detected with a 510±

5 nm detection filter;

(a3);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 530±

5 nm and detected with a 555±

5 nm detection filter;

(a4);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 585±

5 nm and detected with a 610±

5 nm detection filter;

(a5);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 625 ±

10nm and detected with a of 660±

10nm detection filter; and

(a6);

a probe set wherein each probe comprises a fluorophore capable of being excited by an excitation source of 680±

5 nm and detected with a 712 nm long pass detection filter.

27. A kit for detecting a target nucleic acid comprising:

A. at least one hybrid capture probe; and

B. a detection probe set comprising at least four detection probes;

and optionally comprising one or more of the following;

C. a polymerase;

D. a double stranded nucleic acid-specific dye;

E. an anti-DNA-RNA hybrid antibody; and

F. a solid support adapted so as to bind the anti-hybrid antibody;

G. a denaturation reagent;

H. a helicase; and

I. a reagent for fragmenting an amplified nucleic acid.