# US 8,569,019 B2 · Method for performing an assay with a nucleic acid present in a specimen

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- Number: US 8,569,019 B2
- Document ID: US8569019B2
- Status: Granted
- Kind: B2
- Application number: 11/931,722
- Filed: 2007-10-31
- Published: 2008-10-02
- Granted: 2013-10-29
- Earliest claimed: 1998-05-01
- Assignees: Gen-Probe Incorporated
- Applicants: Kelly G. Ammann; Ralph E. Burns; Ernest V. Hansberry; Glenn A. Horner; Cheryl A. Jakub; John E. Kling; Donald J. Nieglos; Robert E. Schneider; Robert J. Smith
- Inventors: Kelly G. Ammann; Ralph E. Burns; Ernest V. Hansberry; Glenn A. Horner; Cheryl A. Jakub; John E. Kling; Donald J. Nieglos; Robert E. Schneider; Robert J. Smith
- Web page: https://patentgrep.com/patent/US8569019B2

## CPC

- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- B01F 29/10: with receptacles rotated about two different axes, e.g. receptacles having planetary motion
- B01F 29/30: Mixing the contents of individual packages or containers, e.g. by rotating tins or bottles
- B01F 29/322: of two or more containers supported for simultaneous mixing, e.g. for bottles in crates
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 7/5255: {by moving sample containers}
- B03C 1/282: {with associated accumulation indicator, e.g. Hall sensor}
- B03C 1/288: {disposed at the outer circumference of a recipient}
- B03C 1/30: Combinations with other devices, not otherwise provided for
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6832: Enhancement of hybridisation reaction
- G01N 35/025: {having a carousel or turntable for reaction cells or cuvettes}
- B01F 29/40354: {arranged for planetary motion}
- B01L 9/06: Test-tube stands; Test-tube holders
- B01L 2200/147: Employing temperature sensors
- B01L 2300/1822: using Peltier elements
- B01L 2300/1827: using resistive heater
- B03C 2201/26: for use in medical or biological applications
- C12Q 2537/10: the purpose or use of
- G01N 35/0098: {involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation (magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C)}
- G01N 35/0099: {comprising robots or similar manipulators (robots per se B25J)}
- G01N 35/1065: {Multiple transfer devices}
- G01N 2035/00356: {Holding samples at elevated temperature (incubation)}
- G01N 2035/00524: {Mixing by agitating sample carrier}
- G01N 2035/0437: {Cleaning cuvettes or reaction vessels}
- G01N 2035/0455: {Coaxial carousels}
- G01N 2035/103: {using disposable tips}
- Y10T 29/53657: to apply or remove a resilient article [e.g., tube, sleeve, etc.]
- Y10T 29/53683: Spreading parts apart or separating them from face to face engagement
- Y10T 29/53843: Tube, sleeve, or ferrule inserting or removing
- Y10T 156/1105: Delaminating process responsive to feed or shape at delamination
- Y10T 156/1906: Delaminating means responsive to feed or shape at delamination
- Y10T 436/11: Automated chemical analysis
- Y10T 436/111666: Utilizing a centrifuge or compartmented rotor
- Y10T 436/113332: with conveyance of sample along a test line in a container or rack
- Y10T 436/114998: with treatment or replacement of aspirator element [e.g., cleaning, etc.]
- Y10T 436/119163: with aspirator of claimed structure
- Y10T 436/2575: Volumetric liquid transfer
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B03C: MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS (separating isotopes B01D59/00; combinations of magnetic or electrostatic separation with separation of solids by other means B03B, B07B)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

An automated process for isolating and amplifying a target nucleic acid in a self-contained analyzer.

## Claims

### Claim 1

A method for detecting the presence of a nucleic acid in a specimen, the method comprising the steps of:
(A) providing a specimen tube containing specimen to a self-contained, standard-alone analyzer;
(B) performing an automated process within a housing of the analyzer that comprises:
(1) transferring at least a portion of the specimen from the specimen tube to a receptacle, wherein the transferred portion of the specimen is dispensed into the receptacle with a robotic pipettor, and wherein the transferred portion of the specimen contains a nucleic acid;
(2) immobilizing the nucleic acid on a solid support within the receptacle, the solid support comprising a magnetically-responsive particle;
(3) applying a magnetic field to the receptacle and its contents;
(4) during step (3), purifying the nucleic acid immobilized in step (2) by accessing and removing at least a portion of a fluid component of the specimen from the receptacle while the nucleic acid remains immobilized on the solid support within the receptacle;
(5) forming a reaction mixture comprising the purified nucleic acid of step (4) and all reagents required to perform a nucleic acid amplification;
(6) performing a nucleic acid amplification with the reaction mixture formed in step (5), the nucleic acid amplification resulting in the enzymatic synthesis of amplification products, each of the amplification products comprising (i) a target sequence contained in the nucleic acid or (ii) the complement of the target sequence;
(7) selectively hybridizing a probe having a label to the target sequence or its complement of an amplification product that is one of the amplification products synthesized in step (6), thereby forming a hybrid in a mixture that comprises the probe and the amplification product; and
(8) in the mixture, detecting [the label as an indication of] the formation of the hybrid, wherein the formation of the hybrid in the mixture is an indication of the presence of the nucleic acid in the specimen.

### Claim 2 (depends on 1)

The method of claim 1 further comprising, prior to step (A), a step of loading the specimen tube onto a specimen tube rack.

### Claim 3 (depends on 1)

The method of claim 1 further comprising, prior to step (A), a step of providing to the analyzer all reagents required to perform a nucleic acid amplification, wherein the specimen tube and the reagents required to perform a nucleic acid amplification are separately provided to the analyzer.

### Claim 4 (depends on 1)

The method of claim 1, wherein the receptacle is a cylindrical tube.

### Claim 5 (depends on 1)

The method of claim 1, wherein the receptacle is one of a plurality of integrally formed receptacles.

### Claim 6 (depends on 5)

The method of claim 5, wherein the integrally formed receptacles are arranged in a linear array.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleic acid is immobilized on the solid support with a capture probe in step (2).

### Claim 8 (depends on 1)

The method of claim 1, wherein the automated process further comprises performing within the housing of the analyzer a step of washing the solid support one or more times after step (4) and prior to step (5).

### Claim 9 (depends on 1)

The method of claim 1, wherein each of the amplification products consists of a nucleic acid sequence having a sequence contained in the nucleic acid or its complement.

### Claim 10 (depends on 1)

The method of claim 1, wherein the amplification products include nucleic acid sequences having a sequence that is the same as the target sequence and nucleic acid and sequences having a sequence that is the complement of the target sequence.

### Claim 11 (depends on 1)

The method of claim 1, wherein step (6) is carried out in a temperature-controlled incubator maintained at a substantially uniform temperature.

### Claim 12 (depends on 1)

The method of claim 1, wherein step (6) is performed in a temperature-controlled chamber of a first incubator, and wherein step (7) is performed in a temperature-controlled chamber of a second incubator, the first and second incubators being in a spaced-apart relationship.

### Claim 13 (depends on 12)

The method of claim 12, wherein the hybrid is in solution.

### Claim 14 (depends on 1)

The method of claim 1, wherein the label is a fluorescent or chemiluminescent label.

### Claim 15 (depends on 1)

The method of claim 1, wherein the housing remains closed during the automated process.

### Claim 16 (depends on 1)

The method of claim 1, wherein the automated process is performed at multiple locations of a processing deck contained within the housing.

### Claim 17

A method for detecting the presence of a nucleic acid in a specimen, the method comprising the steps of:
(A) providing a specimen tube containing a specimen to a self-contained, standard-alone unit analyzer;
(B) performing an automated process within a housing of the analyzer that comprises:
(1) transferring at least a portion of the specimen from the specimen tube to a receptacle, wherein the transferred portion of the specimen is dispensed into the receptacle with a robotic pipettor, and wherein the transferred portion of the specimen contains a nucleic acid;
(2) immobilizing the nucleic acid on a solid support within the receptacle, the solid support comprising a magnetically-responsive particle;
(3) applying a magnetic field to the receptacle and its contents;
(4) during step (3), purifying the nucleic acid immobilized in step (2) by contacting a fluid component of the specimen with a fluid aspirator and aspirating at least a portion of the fluid component from the receptacle while the nucleic acid remains immobilized on the solid support within the receptacle;
(5) forming a reaction mixture comprising the purified nucleic acid of step (4) and all reagents required to perform a nucleic acid amplification;
(6) performing a nucleic acid amplification procedure with the reaction mixture formed in step (5), the nucleic acid amplification resulting in the enzymatic synthesis of amplification products, each of the amplification products comprising (i) a target sequence contained in the nucleic acid or (ii) the complement of the target sequence;
(7) selectively hybridizing a probe having a label to the target sequence or it complement of an amplification product that is one of the amplification products synthesized in step (6), thereby forming a hybrid in a mixture that comprises the probe and the amplification product; and
(8) in the mixture, detecting [the label as an indication of] the formation of the hybrid in the mixture is an indication of the presence of the nucleic acid in the specimen.

### Claim 18 (depends on 17)

The method of claim 17, wherein step (6) is performed in a temperature-controlled chamber of a first incubator, and wherein step (7) is performed in a temperature-controlled chamber of a second incubator, the first and second incubators being in a spaced-apart relationship.

### Claim 19 (depends on 17)

The method of claim 17 further comprising, prior to step (A), a step of loading the specimen tube onto a specimen tube rack.

### Claim 20 (depends on 17)

The method of claim 17 further comprising, prior to step (A), a step of providing to the analyzer all reagents required to perform a nucleic acid amplification, wherein the specimen tube and the reagents required to perform a nucleic acid amplification are separately provided to the analyzer.

### Claim 21 (depends on 17)

The method of claim 17, wherein the receptacle is a cylindrical tube.

### Claim 22 (depends on 17)

The method of claim 17, wherein the receptacle is one of a plurality of integrally formed receptacles.

### Claim 23 (depends on 22)

The method of claim 22, wherein the integrally formed receptacles are arranged in a linear array.

### Claim 24 (depends on 17)

The method of claim 17, wherein the nucleic acid is immobilized on the solid support with a capture probe in step (2).

### Claim 25 (depends on 17)

The method of claim 17, wherein the automated process further comprises performing within the housing of the analyzer a step of washing the solid support one or more times after step (4) and prior to step (5).

### Claim 26 (depends on 17)

The method of claim 17, wherein the fluid aspirator comprises an aspirator tube in frictional engagement with a contamination-limiting element configured to permit the passage of the fluid component of the specimen therethrough, and wherein the contamination-limiting element contacts the fluid component of the specimen in step (4).

### Claim 27 (depends on 26)

The method of claim 26, wherein the contamination-limiting element is a tiplet.

### Claim 28 (depends on 17)

The method of claim 17, wherein each of the amplification products consists of a nucleic acid sequence having the sequence contained in the nucleic acid or its complement.

### Claim 29 (depends on 17)

The method of claim 17, wherein the amplification products include nucleic acid sequences having a sequence that is the same as the target sequence and nucleic acid sequences having a sequence that is the complement of the target sequence.

### Claim 30 (depends on 17)

The method of claim 17, wherein step (6) is carried out in a temperature-controlled incubator maintained at a substantially uniform temperature.

### Claim 31 (depends on 17)

The method of claim 17, wherein the label is a fluorescent or chemiluminescent label.

### Claim 32 (depends on 17)

The method of claim 17, wherein the housing remains closed during the automated process.

### Claim 33 (depends on 17)

The method of claim 17, wherein the automated process is performed at multiple locations of a processing deck contained within the housing.

### Claim 34 (depends on 17)

The method of claim 17, wherein the hybrid is in solution.