Molecular diagnostics system and methods

• US 9,752,182 B2
• Filed: 08/28/2014
• Issued: 09/05/2017
• Est. Priority Date: 12/23/2004
• Status: Expired due to Fees

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Inventors: Collier, Gordon Bruce, Wood, John Allister, MacLeod, Jason Andrew, Dicke, William Charles, Nemeth, Attila Csaba, Miller, Cary James
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/471,166
• Publication Number:
  US 20150064707A1
• Time in Patent Office: 1,104 Days
• Field of Search: 435 61, 435 911, 435 612, 435 912
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502769 characterised by multiphase flow arrangements
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/0335 using coils
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical or biological applications
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]

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

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.

2. 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.

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

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