# US 9,752,182 B2 · Molecular diagnostics system and methods

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- Number: US 9,752,182 B2
- Document ID: US9752182B2
- Status: Granted
- Kind: B2
- Application number: 14/471,166
- Filed: 2014-08-28
- Published: 2015-03-05
- Granted: 2017-09-05
- Earliest claimed: 2004-12-23
- Assignees: Abbott Point of Care Inc.
- Applicants: Abbott Point of Care Inc.
- Inventors: Gordon Bruce Collier; John Allister Wood; Jason Andrew MacLeod; William Charles Dicke; Attila Csaba Nemeth; Cary James Miller
- Web page: https://patentgrep.com/patent/US9752182B2

## CPC

- C12Q 1/6853: using modified primers or templates
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- B01L 3/502769: {characterised by multiphase flow arrangements}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B03C 1/01: by addition of magnetic adjuvants
- B03C 1/0332: {using permanent magnets}
- B03C 1/0335: {using coils}
- B03C 1/288: {disposed at the outer circumference of a recipient}
- B03C 1/30: Combinations with other devices, not otherwise provided for
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/686: Polymerase chain reaction [PCR]
- B01L 2200/027: for microfluidic devices
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2400/0421: electrophoretic flow
- B01L 2400/0487: fluid pressure, pneumatics
- B03C 2201/18: Magnetic separation whereby the particles are suspended in a liquid
- B03C 2201/26: for use in medical or biological applications
- C12N 15/1013: {by using magnetic beads}
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. In one embodiment, a sample comprising cells containing nucleic acid is exposed to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells to form a nucleic acid-bead complex. The nucleic acid-bead complex is passed through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture. The one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field. The invention is particularly suited for use in point-of-care medical diagnostics testing.

## Claims

### Claim 1

A method for performing a nucleic acid amplification assay comprising:
(a) combining reagents, DNA polymerase, a target nucleic acid and an amount of a first modified primer comprising a sequence of bases complementary to a first region of the target nucleic acid, a polymerase blocking region attached to the first modified primer, a first single stranded hybridization region attached to the polymerase blocking region, and a detectable label,
(b) cycling a mixture of (a) to provide multiple copies of an amplicon incorporating the first modified primer;
(c) substantially eliminating excess unincorporated first modified primer from the mixture;
(d) exposing the mixture to a capture oligonucleotide complementary to the first single stranded hybridization region;
(e) hybridizing the first single stranded hybridization region of the amplicon incorporating the first modified primer, with the capture oligonucleotide; and
(f) detecting the label associated with the hybridization.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
the combining further comprises combining an amount of a second modified primer comprising a sequence of bases complementary to a second region of the target nucleic acid, a polymerase blocking region attached to the second modified primer, a second single stranded hybridization region attached to the polymerase blocking region, and a detectable label;
the cycling further comprises cycling a mixture of (a) to provide multiple copies of an amplicon incorporating the first modified primer and the second modified primer;
the substantially eliminating further comprises the substantially eliminating excess unincorporated second modified primer from the mixture;
exposing the mixture further comprises exposing the mixture to a capture oligonucleotide complementary to both of the first single stranded hybridization region and the second single stranded hybridization region; and
hybridizing further comprises hybridizing both of the first single stranded hybridization region and the second single stranded hybridization region of the amplicon incorporating the first modified primer and the second modified primer, with the capture oligonucleotide.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polymerase blocking region is phosphoraramidite 18-O-dimethoxyltritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

### Claim 4 (depends on 1)

The method of claim 1, wherein the delectable label is biotin, FAM, or fluorocein.