Nucleic acid assays

• US 20030077580A1
• Filed: 08/08/2002
• Published: 04/24/2003
• Est. Priority Date: 05/02/1997
• Status: Active Grant

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux Vitek
• Inventors: Moe, James G., Catanzariti, Luigi, Kluttz, Bryan W., McKinley, Geoff A., Burg, J. Lawrence, Vera-Garcia, Marcela
• Granted Patent: US 7,262,008 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes
• CPC Class Codes: G01N 35/026 having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to the detection of specific nucleic acid sequences, either by a process of amplification of specific nucleic acid sequences or not. More particularly the invention provides for improved compositions and methods for reducing the chance for contamination from manipulation of reagents, internal controls for amplification, and the use of automated apparatus for the automated detection of one, or more than one amplified nucleic acid sequences.

Claims

1. A method for the specific automated detection of one or more viral or microorganism nucleic acids in a sample, said method comprising:

a) lysing at least one microorganism in said sample, if present, to liberate target nucleic acid;

b) amplifying said nucleic acid to form amplicons;

c) contacting said sample with a solid phase receptacle coated with a capture nucleic acid, wherein said capture nucleic acid can form a hybridization complex with said amplicons;

d) allowing said hybridization complex to form;

e) contacting said hybridization complex to a detection nucleic acid, wherein said detection nucleic acid can form a specific hybridization detection complex with said amplicon; and

wherein the detection of nucleic acid is conjugated to a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore;

f) allowing said detection complex to form;

g) detecting said detectable signal if said amplicon is present in said sample;

h) wherein optionally, between step (d) and (e) or (f) and (g), said hybridization complex can be washed to remove excess non-specifically bound nucleic acid.

2. The method of claim 1, wherein said microorganism or virus is selected from the group consisting of Mycobacterium tuberculosis (M.tb), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Human Immunodeficiency Virus (HIV).

3. A device for the automated detection of a first target nucleic acid and a second target nucleic acid, said apparatus comprising a solid phase receptacle, wherein said receptacle is coated with a first capture nucleic acid which can form a specific hybridization complex with said first target nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second target nucleic acid.

4. The device of claim 3 wherein said first capture nucleic acid and said second capture nucleic acid are coated on said solid phase receptacle in two distinct zones.

5. The device of claim 3 wherein first capture nucleic acid and said second capture nucleic acid are coated on said solid phase receptacle in a single zone.

6. The device of claim 3, modified for the detection of more than two nucleic acids, wherein the device further comprises additional capture nucleic acids which can each form a specific hybridization complex with additional target nucleic acid other than said first or second target nucleic acid in said sample.

7. A method for the automated detection of the presence or absence of a first target nucleic acid and a second target nucleic acid in a sample, said method comprising:

a) contacting said sample with a solid phase receptacle, wherein said receptacle is coated with a first capture nucleic acid which can form a specific hybridization complex with said first target nucleic acid, and a second capture acid which can form a specific hybridization complex with said second target nucleic acid;

b) allowing specific hybridization complexes to form if said nucleic acid is present;

c) contacting said solid phase receptacle hybridization complex with a first detection nucleic acid, wherein said first detection nucleic acid can form a specific hybridization detection complex with said first target nucleic acid, and wherein said first detection nucleic acid is conjugated to a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore;

d) allowing specific detection complexes to form;

e) detecting said signal if said first target nucleic acid is in said sample;

f) contacting said solid phase receptacle hybridization complex with a second detection nucleic acid, wherein said second detection nucleic acid can form a specific hybridization detection complex with said second target nucleic acid, and wherein said second detection nucleic acid is conjugated a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore;

g) allowing specific detection complexes to form;

h) detecting said signal if said second target nucleic acid is in said sample;

i) and wherein optionally, between steps (b) and (c), (d) and (e) or (g) and (h) hybridization complex can be washed to remove excess non-specifically bound nucleic acid;

j) wherein the absence of a detectable signal correlates with the absence of said nucleic acid in said sample and the presence of a signal in step (e) or step (h) indicates the presence of said first target nucleic acid or the presence of said second target nucleic acid, respectively.

8. The method of claim 7 which further incorporates internal control molecules.

9. The method of claim 7 which further includes amplification of a target nucleic acid before detection of the target nucleic acid.

10. The method of claim 7, which further comprises detection of primer control sequences.

11. The method of claim 7, wherein one or more of said steps (c) and (f) are performed concurrently, said steps (d) and (g) are performed concurrently, and steps (e) and (h) are performed concurrently.

12. The method of claim 7, wherein said detectable signal is alkaline phosphate, which is detected with a methyl umbelliferyl phosphate substrate.

13. The method of claim 7, modified for the detection of more than two nucleic acids, wherein said solid phase receptacle is further coated with one or more additional specific capture nucleic acids, wherein said additional specific capture nucleic acids can form specific hybridization complexes with one or more additional target nucleic acids other than said first and second target nucleic acids in said sample.

14. The method of claim 7 wherein said first target nucleic acid is specific for Chlamydia trachomatis (CT), and said second target nucleic acid is specific for Neisseria gonorrhoeae (NG).

15. The method of claim 13 wherein said first target nucleic acid is specific for Chlamydia trachomatis (CT), and said second target nucleic acid is specific for Neisseria gonorrhoeae (NG).