# US 10,246,737 B2 · Methods for detecting multiple nucleic acids in a sample using reporter compounds and binding members thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,737 B2 - Document ID: US10246737B2 - Status: Granted - Kind: B2 - Application number: 15/116,185 - Filed: 2015-02-23 - Published: 2017-03-23 - Granted: 2019-04-02 - Earliest claimed: 2014-02-21 - Assignees: Alere Technologies GmbH - Applicants: Alere Technologies GmbH - Inventors: Thomas Ullrich - Web page: https://patentgrep.com/patent/US10246737B2 ## CPC - C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer - B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces} - C12Q 1/6844: Nucleic acid amplification reactions - G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} - G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching - B01L 2300/0819: Microarrays; Biochips - B01L 2400/0481: squeezing of channels or chambers - G01N 21/645: {Specially adapted constructive features of fluorimeters} - 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 - 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) ## Abstract The present invention relates to methods for determining the presence and/or amount of multiple nucleic acids in a sample. ## Claims ### Claim 1 A method of determining the presence and/or amount of nucleic acids in a sample, comprising (a) introducing into a reaction chamber an amount of a first nucleic acid; an amount of a second nucleic acid; an amount of a FRET compound capable of forming complexes with the first nucleic acid, wherein the FRET compound comprises a binding portion specific to a region of the first nucleic acid, and a first detectable label that has, in an uncomplexed state, a first detectable characteristic and, in a complexed state, a second detectable characteristic; an amount of a reporter compound capable of forming complexes with the second nucleic acid, wherein each reporter compound comprises a binding portion specific to a region of the second nucleic acid and a second detectable label, wherein said second detectable label is identical to said first detectable label; and wherein the reaction chamber comprises a binding member capable of capturing the reporter compound; wherein forming of complexes of the second nucleic acid with the reporter compound inhibits capturing of the reporter compound by the binding member; (b) subjecting the first and/or second nucleic acid to amplification; (c) forming complexes each comprising a first nucleic acid and a FRET compound; (d) forming complexes of a subset of the amount of reporter compound with a second nucleic acid; (e) capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member; (f) determining a value indicative for the presence and/or amount of first nucleic acid in complex with FRET compounds; and (g) determining a value indicative for the presence and/or amount of reporter compound captured on the binding member. ### Claim 2 (depends on 1) The method of claim 1, further comprising determining a value indicative for the presence and/or amount of second nucleic acid based on the value indicative for the presence and/or amount of reporter compound captured on the binding member. ### Claim 3 (depends on 1) The method of claim 1, wherein the first detectable label is a fluorescent label. ### Claim 4 (depends on 1) The method of claim 1, wherein the second detectable label is a fluorescent label. ### Claim 5 (depends on 1) The method of claim 1, wherein both the first detectable label and the second detectable label are fluorescent labels. ### Claim 6 (depends on 5) The method of claim 5, wherein both the first detectable label and the second detectable label emit in a wavelength range of from 300 nm to 850 nm. ### Claim 7 (depends on 1) The method of claim 1, wherein the value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds is determined in a complexed state of the FRET compounds. ### Claim 8 (depends on 1) The method of claim 1, wherein the first nucleic acid is an analyte. ### Claim 9 (depends on 1) The method of claim 1, wherein the second nucleic acid is selected from the group consisting of a second analyte and a control nucleic acid. ### Claim 10 (depends on 1) The method of claim 1, wherein the first nucleic acid is present in an amount of less than 100 copies per sample. ### Claim 11 (depends on 1) The method of claim 1, wherein the second nucleic acid is present in an amount of 100 copies or more per sample. ### Claim 12 (depends on 1) The method of claim 1, wherein the steps of (i) forming complexes each comprising a first nucleic acid and a FRET compound, (ii) forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of second nucleic acid and (iii) capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member are performed concomitantly. ### Claim 13 (depends on 1) The method of claim 1, wherein amplification of the first and second nucleic acid is initiated prior to the step of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of second nucleic acid. ### Claim 14 (depends on 1) The method of claim 1, wherein the steps of (i) forming complexes each comprising a first nucleic acid and a FRET compound, (ii) forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of second nucleic acid, (iii) capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member, and (iv) subjecting the first and/or second nucleic acid to amplification are performed concomitantly. ### Claim 15 (depends on 13) The method of claim 13, wherein the second nucleic acid is selected from the group consisting of a positive amplification control and a negative amplification control. ### Claim 16 (depends on 1) The method of claim 1, wherein the binding member comprises one or more different reporter specific capture compounds being capable of capturing a reporter compound on the binding member. ### Claim 17 (depends on 16) The method of claim 16, wherein the reporter specific capture compounds are oligonucleotides. ### Claim 18 (depends on 16) The method of claim 16, wherein the different reporter specific capture compounds are arranged on different locations with respect to the binding member. ### Claim 19 (depends on 16) The method of claim 16, wherein the reporter compounds are captured on the binding member by forming complexes with the reporter specific capture compounds. ### Claim 20 (depends on 16) The method of claim 16, wherein at least a part of an interaction site of the reporter compound being capable of forming a complex with a second nucleic acid is also capable of forming a complex with a reporter specific capture compound. ### Claim 21 (depends on 16) The method of claim 16, wherein the reporter specific capture compounds and the second nucleic acid compete for forming a complex with the reporter compound. ### Claim 22 (depends on 1) The method of claim 1, wherein the value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds and/or the value indicative for the presence and/or amount of reporter compound captured on the binding member is determined in a reaction chamber comprising the binding member and a cover element, wherein optionally the cover element is configured to be at least partially deformable such that the reaction chamber has a relaxed state interior volume and a compressed state interior volume. ### Claim 23 (depends on 1) The method of claim 1, wherein the value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds and/or the value indicative for the presence and/or amount of reporter compound captured on the binding member is determined in a reaction chamber comprising a first surface, a second surface and the binding member capable of capturing the reporter compound, wherein the distance between the first and the second surface can be varied such that the reaction chamber has a relaxed state interior volume and a compressed state interior volume. ### Claim 24 (depends on 23) The method of claim 23, wherein the value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds is determined in the reaction chamber having a relaxed state interior volume. ### Claim 25 (depends on 22) The method of claim 22, wherein the value indicative for the presence and/or amount of reporter compound captured on the binding member is determined in the reaction chamber having a compressed state interior volume. ### Claim 26 (depends on 1) The method of claim 1, wherein the value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds and the value indicative for the presence and/or amount of reporter compound captured on the binding member are determined consecutively or concomitantly. ### Claim 27 (depends on 1) The method of claim 1, wherein the reporter compounds are oligonucleotides. ### Claim 28 (depends on 1) The method of claim 1, further comprising subjecting the first and/or second nucleic acids to reverse transcription prior to subjecting them to amplification. ### Claim 29 (depends on 13) The method of claim 13, wherein the amplification comprises a step of denaturing double stranded nucleic acids, wherein the double stranded nucleic acids comprise complexes of reporter compounds with second nucleic acids, complexes of reporter compounds with reporter specific capture compounds, double strands of reporter compounds and double strands of first and/or second nucleic acids, and/or wherein the amplification comprises a step of annealing primer compounds to first and/or second nucleic acids, wherein optionally the annealing step is performed concomitantly with the step of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of second nucleic acid and/or the step of capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member. ### Claim 30 (depends on 13) The method of any of claim 13, wherein the amplification is an isothermal amplification method, wherein the isothermal amplification method optionally is an isothermal NEAR. ### Claim 31 (depends on 13) The method of claim 13, wherein the amplification is a cyclic amplification, wherein the cyclic amplification optionally is a PCR, wherein performing the PCR optionally comprises using a polymerase having exonuclease activity, wherein optionally the value indicative for the presence and/or amount of reporter compound captured on the binding member is determined after at least one cycle of the cyclic amplification, and optionally after each cycle of the cyclic amplification, wherein optionally the value indicative for the presence and/or amount of first and/or second nucleic acid is determined each time after determining the value indicative for the presence and/or amount of reporter compound captured on the binding member. ### Claim 32 (depends on 2) The method of claim 2, wherein the value indicative for the presence and/or amount of second nucleic acid is determined based on a calibration curve correlating the value indicative for the presence and/or amount of reporter compound with the value indicative for the presence and/or amount of second nucleic acid. ### Claim 33 A method of determining the presence and/or amount of nucleic acids in a sample, comprising providing in a reaction chamber an amount of a first nucleic acid; an amount of a second nucleic acid; an amount of a FRET compound capable of forming complexes with the first nucleic acid, wherein the FRET compound comprises a binding portion specific to a region of the first nucleic acid, and a first detectable label that has, in an uncomplexed state, a first detectable characteristic and, in a complexed state, a second detectable characteristic; and an amount of a reporter compound capable of forming complexes with the second nucleic acid, wherein each reporter compound comprises a binding portion specific to a region of the second nucleic acid and a second detectable label, wherein said second detectable label is identical to said first detectable label; wherein the reaction chamber comprises a binding member capable of capturing the reporter compound, wherein forming of complexes of the second nucleic acid with the reporter compound inhibits capturing of the reporter compound by the binding member, and a cover element which is configured to be at least partially deformable such that the reaction chamber has a relaxed state interior volume and a compressed state interior volume; subjecting the first and/or second nucleic acid to an isothermal amplification; forming complexes each comprising a first nucleic acid and a FRET compound; forming complexes of a subset of the amount of reporter compound with a second nucleic acid; capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member; determining a value indicative for the presence and/or amount of first nucleic acid in complex with FRET compounds in solution in the reaction chamber having a relaxed state interior volume; and determining a value indicative for the presence and/or amount of reporter compound captured on the binding member in the reaction chamber having a compressed state interior volume, and, based on the value indicative for the presence and/or amount of reporter compound captured on the binding member, determining a value indicative for the presence and/or amount of second nucleic acid. ### Claim 34 A method of determining the presence and/or amount of nucleic acids in a sample, comprising introducing into a reaction chamber an amount of a first nucleic acid; an amount of a second nucleic acid; an amount of a FRET compound capable of forming complexes with the first nucleic acid, wherein the FRET compound comprises a binding portion specific to a region of the first nucleic acid, and a first detectable label that has, in an uncomplexed state, a first detectable characteristic and, in a complexed state, a second detectable characteristic; and an amount of a reporter compound capable of forming complexes with the second nucleic acid, wherein each reporter compound comprises a binding portion specific to a region of the second nucleic acid and a second detectable label, wherein said second detectable label is identical to said first detectable label; wherein the reaction chamber comprises a first surface, a second surface and a binding member capable of capturing the reporter compound, wherein the distance between the first and the second surface can be varied such that the reaction chamber has a relaxed state interior volume and a compressed state interior volume and wherein forming of complexes of the second nucleic acid with the reporter compound inhibits capturing of the reporter compound by the binding member; subjecting the first and/or second nucleic acid to an isothermal amplification; forming complexes each comprising a first nucleic acid and a FRET compound; forming complexes of a subset of the amount of reporter compound with a second nucleic acid; capturing a remaining subset of the amount of reporter compound not in complex with a second nucleic acid on the binding member; determining a value indicative for the presence and/or amount of first nucleic acid captured by FRET compounds in solution in the reaction chamber having a relaxed state interior volume; and determining a value indicative for the presence and/or amount of reporter compound captured on the binding member in the reaction chamber having a compressed state interior volume, and, based on the value indicative for the presence and/or amount of reporter compound captured on the binding member, determining a value indicative for the presence and/or amount of second nucleic acid.