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WO2025202488A1 - Activateur d'amplification d'acide nucléique - Google Patents

Activateur d'amplification d'acide nucléique

Info

Publication number
WO2025202488A1
WO2025202488A1 PCT/EP2025/058616 EP2025058616W WO2025202488A1 WO 2025202488 A1 WO2025202488 A1 WO 2025202488A1 EP 2025058616 W EP2025058616 W EP 2025058616W WO 2025202488 A1 WO2025202488 A1 WO 2025202488A1
Authority
WO
WIPO (PCT)
Prior art keywords
amplification
halogen
nucleic acid
enhancer
methyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/058616
Other languages
English (en)
Inventor
Martin REMPT
Sheila ISEPPONI
Erdmann SCHEFFER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics International AG
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics International AG
Roche Diagnostics GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics International AG, Roche Diagnostics GmbH filed Critical Roche Diagnostics International AG
Publication of WO2025202488A1 publication Critical patent/WO2025202488A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Bovine Serum Albumin a protein that can reduce nonspecific binding of DNA polymerase to other components in the reaction or to walls of the reaction tube and may reduce inhibition of RT-PCR by melanin, which can improve PCR specificity and yield (Giambernardi et al., Biotechniques 25, 564-6.); DMSO (dimethyl sulfoxide): a solvent that can help denature secondary structures in DNA templates, reduce PCR inhibitors, and improve PCR yield, but which may also act as an inhibitor of DNA polymerases in higher concentrations (Varadaraj and Skinner, (1994) Gene 140, 1-5); Betaine: a chemical that can reduce the formation of secondary structures in DNA templates and improve PCR efficiency, especially for GC-rich templates (Rees et al.
  • BSA Bovine Serum Albumin
  • Tween® 20, NP-40 and Triton® X-100 non-ionic detergents that can improve PCR sensitivity by reducing the binding of PCR inhibitors to the reaction components thereby overcoming inhibitory effects even of trace amounts of strong ionic detergents such as, e.g., SDS (Gelfand et al.
  • PCR Protocols A Guide to Methods and Applications, eds, Academic Press, San Diego, CA, 129-41
  • Formamide a denaturant that can help reduce secondary structures in DNA templates, reduce the melting temperature of DNA, and improve PCR yield in particular in GC-rich templates, but which may also act as an inhibitor of DNA polymerases in higher concentrations
  • TaqStart Antibody an antibody that can bind to and inhibit non-specific amplification by Taq-derived DNA polymerases, which can improve PCR specificity in hot start PCR applications (Kellogg, et al. (1994) BioTechniques, 16, 1134- 1137).
  • R 1 CH 3 , H, CH2CH3 or CH2CHOHCH2OH
  • R 3 OH, H, CH3, CH2/halogen, CH/halogem, C/halogen 3 or a halogen
  • R 4 H, OH, CH3, CH2/halogen, CH/halogem, C/halogen 3 or a halogen.
  • R 3 and R 4 may be OH in the same amplification enhancer structure (i.e., either R 3 is OH, while R 4 is a different residue selected from the list for R 4 (i.e., not OH) or R 4 is OH, while R 3 is a different residue selected from the list for R 3 (i.e., not OH)) and only either one of R 3 and R 4 may be H in the same amplification enhancer structure if R 2 is NH 2 (i.e., if R 2 is NH 2 either R 3 is H, while R 4 is a different residue selected from the list for R 4 (i.e., not H) or R 4 is H, while R 3 is a different residue selected from the list for R 3 (i.e., not H)).
  • R 3 and R 4 may be OH in the same amplification enhancer structure (i.e., either R 3 is OH, while R 4 is a different residue selected from the list for R 4 (i.e., not OH) or R 4 is OH, while R 3 is a different residue selected from the list for R 3 (i.e., not OH)) and only either one of R 3 and R 4 may be H in the same amplification enhancer structure if R 2 is NH 2 (i.e., if R 2 is NH 2 either R 3 is H, while R 4 is a different residue selected from the list for R 4 (i.e., not H) or R 4 is H, while R 3 is a different residue selected from the list for R 3 (i.e., not H)).
  • the method further comprises:
  • step (b) performing a hybridizing step, wherein the hybridizing step comprises contacting the amplification product from step (a) with one or more probes;
  • the amplification enhancer is present in step (a) at a concentration of 0.005% to 0.1% (m/V), preferably 0.01% to 0.09% (m/V), such as 0.09% (m/V).
  • R 3 and R 4 may be OH in the same amplification enhancer structure (i.e., either R 3 is OH, while R 4 is a different residue selected from the list for R 4 (i.e., not OH) or R 4 is OH, while R 3 is a different residue selected from the list for R 3 (i.e., not OH)) and only either one of R 3 and R 4 may be H in the same amplification enhancer structure if R 2 is NH2 (i.e., if R 2 is NH2 either R 3 is H, while R 4 is a different residue selected from the list for R 4 (i.e., not H) or R 4 is H, while R 3 is a different residue selected from the list for R 3 (i.e., not H)).
  • the step of amplifying the nucleic acid comprises contacting the nucleic acid with a polymerase, dNTPs, and one or more set of primers specific for the nucleic acid.
  • the amplification enhancer is present at a concentration of 0.005% to 0.1% (m/V), preferably 0.01% to 0.09% (m/V), such as 0.09% (m/V), in the step of amplifying the nucleic acid.
  • the presence of the amplification enhancer results in a more efficient amplification of the nucleic acid compared to the amplification under the same conditions but without the amplification enhancer.
  • kits for amplifying a nucleic acid comprising an amplification enhancer as disclosed herein.
  • the kit for amplifying a nucleic acid comprises:
  • R 3 and R 4 may be OH in the same amplification enhancer structure (i.e., either R 3 is OH, while R 4 is a different residue selected from the list for R 4 (i.e., not OH) or R 4 is OH, while R 3 is a different residue selected from the list for R 3 (i.e., not OH)) and only either one of R 3 and R 4 may be H in the same amplification enhancer structure if R 2 is NH2 (i.e., if R 2 is NH2 either R 3 is H, while R 4 is a different residue selected from the list for R 4 (i.e., not H) or R 4 is H, while R 3 is a different residue selected from the list for R 3 (i.e., not H)).
  • the amplification enhancer is comprised in a composition configured to provide a final concentration of the amplification enhancer of 0.005% to 0.1% (m/V), preferably 0.01% to 0.09% (m/V), such as 0.09% (m/V), during the amplification of the nucleic acid.
  • the disclosure also provides a reaction mixture for amplifying a nucleic acid, the reaction mixture comprising the amplification enhancer disclosed herein.
  • the reaction mixture for amplifying a nucleic acid comprises:
  • R 3 and R 4 may be OH in the same amplification enhancer structure (i.e., either R 3 is OH, while R 4 is a different residue selected from the list for R 4 (i.e., not OH) or R 4 is OH, while R 3 is a different residue selected from the list for R 3 (i.e., not OH)) and only either one of R 3 and R 4 may be H in the same amplification enhancer structure if R 2 is NH2 (i.e., if R 2 is NH2 either R 3 is H, while R 4 is a different residue selected from the list for R 4 (i.e., not H) or R 4 is H, while R 3 is a different residue selected from the list for R 3 (i.e., not H)).
  • the amplification enhancer is present at a concentration of 0.005% to 0.1% (m/V), preferably 0.01% to 0.09% (m/V), such as 0.09% (m/V).
  • the polymerase is not particularly limited, including a DNA polymerase, a RNA polymerase or a reverse transcriptase. In some embodiments, the polymerase is a DNA polymerase.
  • the dNTPs are not particularly limited, including naturally occurring nucleotides and modified nucleotides. Naturally occurring nucleotides include, for example, dATP, dTTP, dCTP and/or dGTP.
  • the type of nucleic acid is not particularly limited, but will typically be an RNA or a DNA, preferably a DNA.
  • the nucleic acid can be comprised in a biological sample or non-biological sample, preferably in a biological sample.
  • the amplification enhancer is selected from methylparaben, 4- hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2- hydroxybenzoate, methyl-4-bromo-2-hydroxybenzoate, 4-hydroxy-2-methylbenzoic acid, 4- (methylamine)benzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-salicylic acid, 2-Fluoro-4- hydroxybenzoic acid, 4-Hydroxy-2-(trifluoromethyl)benzoic acid, Arm-43, Methyl 4-hydroxy- 2-methylbenzoate, and ethylparaben.
  • the amplification enhancer is not methylparaben.
  • the amplification enhancer is selected from 4-hydroxybenzoic acid, methyl-2,4- dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl-4-bromo-2- hydroxybenzoate, 4-hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-2- methylbenzoic acid, 4-amino-salicylic acid, 2-Fluoro-4-hydroxybenzoic acid, 4-Hydroxy-2- (trifluoromethyl)benzoic acid, Arm-43, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben, 4- Hydroxy-N,N-dimethylbenzamide, 4-Acetamidophenol, and thymol.
  • the amplification enhancer is selected from 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl-4-bromo-2-hydroxybenzoate, 4- hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-2-methylbenzoic acid, 4- amino-salicylic acid, 2-Fluoro-4-hydroxybenzoic acid, 4-Hydroxy-2-(trifluoromethyl)benzoic acid, Arm-43, Methyl 4-hydroxy-2-m ethylbenzoate, and ethylparaben.
  • the amplification enhancer is selected from methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben and thymol. In some embodiments, the amplification enhancer is selected from, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2- methylbenzoate, and ethylparaben. In some embodiments, the amplification enhancer is selected from methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2-methylbenzoate, and ethylparaben.
  • the amplification enhancer is a mixture of two or more enhancers selected from the group consisting of methylparaben, 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl-4-bromo-2-hydroxybenzoate, 4-hydroxy-2-methyl- benzoic acid, 4-(methylamine)benzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-salicylic acid, 2-Fluoro-4-hydroxybenzoic acid, 4-Hydroxy-2-(trifluoromethyl)benzoic acid, Arm -43, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben, 4-Hydroxy-N,N-dimethylbenzamide, 4- Acetamidophenol, and thymol.
  • the amplification enhancer is a mixture of two or more enhancers selected from the group consisting of methylparaben, 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl-4-bromo-2-hydroxybenzoate, 4-hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-salicylic acid, 2-Fluoro-4-hydroxybenzoic acid, 4- Hydroxy-2-(trifluoromethyl)benzoic acid, Arm-43, Methyl 4-hydroxy-2-m ethylbenzoate, and ethylparaben.
  • methylparaben 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl-4-bromo-2-hydroxybenz
  • the amplification enhancer is a mixture of two or more enhancers selected from consisting of from methylparaben, 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, 4- hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-salicylic acid, Arm -43, Methyl 4-hydroxy-2-methylbenzoate, and ethylparaben.
  • the amplification enhancer is a mixture of two or more enhancers selected from consisting of methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben and thymol.
  • the amplification enhancer is a mixture of two or more enhancers selected from consisting of methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2- methylbenzoate, and ethylparaben.
  • the amplification enhancer is a mixture of two or more enhancers selected from the group consisting of 4-hydroxybenzoic acid, methyl-2,4-dihydroxybenzoate, methyl-p-anisate, methyl-4-fluoro-2-hydroxybenzoate, methyl- 4-bromo-2-hydroxybenzoate, 4-hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-salicylic acid, 2-Fluoro-4-hydroxybenzoic acid, 4- Hydroxy-2-(trifluoromethyl)benzoic acid, Arm-43, Methyl 4-hydroxy-2-m ethylbenzoate, and ethylparaben.
  • each of the enhancers may present at a concentration of 0.005% to 0.1% (m/V), preferably 0.01% to 0.09% (m/V), such as 0.09% (m/V).
  • a method for amplifying a nucleic acid comprising:
  • step (b) performing a hybridizing step, wherein the hybridizing step comprises contacting the amplification product from step (a) with one or more probes;
  • dNTPs comprise dATP, dTTP, dCTP, and dGTP.
  • nucleic acid is an RNA or a DNA.
  • nucleic acid is comprised in a biological sample.
  • amplification enhancer is selected from methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2- methylbenzoate, ethylparaben and thymol or any combination thereof.
  • step (a) The method of any one of the preceding items, wherein the amplification enhancer is present in step (a) at a concentration of 0.01% to 0.09% (m/V).
  • kits of item 17 or 18, wherein the dNTPs comprise dATP, dTTP, dCTP, and dGTP.
  • nucleic acid is an RNA or a DNA.
  • kits of item 21, wherein the biological sample is a bodily sample, such as blood, plasma, or urine.
  • kits of any one of items 17-26, wherein the amplification enhancer is selected from methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben and thymol or any combination thereof.
  • kits 17-28 The kit of any one of items 17-28, wherein the amplification enhancer is a mixture of methylparaben and methyl-2,4-dihydroxy benzoate.
  • step of amplifying the nucleic acid comprises contacting the nucleic acid with a polymerase, dNTPs, and one or more set of primers specific for the nucleic acid.
  • dNTPs comprise dATP, dTTP, dCTP, and dGTP.
  • reaction mixture of any one of items 47-49, wherein the nucleic acid is an RNA or a DNA.
  • the reaction mixture of any one of items 47-50, wherein the reaction mix is for amplifying a nucleic acid comprised in a biological sample.
  • reaction mixture of item 51 wherein the biological sample is a bodily sample, such as blood, plasma, or urine.
  • reaction mixture of any one of items 47-56, wherein the amplification enhancer is selected from methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2- methylbenzoate, ethylparaben and thymol or any combination thereof.
  • Figure 1 shows an overview over the experimental design for example 1 and example 2.
  • Figure 2 shows results from one-factorial screening tests.
  • the top left panel shows a boxplot for Delta Ct when using one of compounds 1, 3 or 5 vs. baseline (BSL) for HIV ssRNA as template.
  • the bottom left panel shows a boxplot for Delta Ct when using one of compounds 1, 3 or 5 vs. baseline (BSL) for HBV dsDNA as template.
  • the top right panel shows a boxplot for Delta Ct when using one of compounds 1, 15, 16 or 1 and 5 in combination vs. baseline (BSL) for HIV ssRNA as template.
  • the bottom right panel shows a boxplot for Delta Ct when using one of compounds 1, 15, 16 alone or 1 and 5 in combination (“Cln5) vs. baseline (BSL) for HBV dsDNA as template.
  • Figure 3 shows results from two-factorial tests.
  • the left panel shows a boxplot for Delta Ct when using one of compounds 1, 3, 5, 15, 16 alone or 1 and 5 in combination (“C01n05”) vs. baseline (BSL) for HIV ssRNA as template.
  • the right panel shows a boxplot for Delta Ct when using one of compounds 1, 3, 5, 15, 16 alone or 1 and 5 in combination (“C01n05”) vs. baseline (BSL) for HBV dsDNA as template.
  • Figure 4 shows results from two-factorial tests for combination of compound 1 and 5.
  • the left panel shows Ct values when using one of compounds 1 or 5 alone or in combination (“C01n05”) or baseline (BSL) in the presence of three different amplification inhibitors for HIV ssRNA as template.
  • the right panel shows Ct values when using one of compounds lor 5 alone or in combination (“C01n05”) or baseline (BSL) in the presence of three different amplification inhibitors for HBV dsDNA as template.
  • Figure 5 shows the experimental design for a one-factorial test using Taq Polymerase for Norro GG2 ssRNA as template.
  • Figure 6 Box Plots show the CP results for a one-factorial test using Taq Polymerase for Norro GG2 ssRNA as template over three different titer parts.
  • the left panel shows baseline performance of taq assay, no enhancer resulting in adequate Total Gage R&R.
  • the right panel shows the effect of combination of compound 1 and 5 demonstrating desirable total Gage R&R and therefore decreased variation by repeatability and reproducibility
  • Figure 7 shows a Bar Chart of Mean Delta Ct for the data from Example 1 for compounds 1, 3, 5, 15, 16 alone or 1 and 5 in combination (“CO ln05”) vs. baseline for both HIV, ssRNA (Channel 2) and HBV dsDNA (Channel 3) as template.
  • Figure 8 shows a Bar Chart of Mean Delta Ct for the data from Example 2 for compounds 1, 3, 5, 15, 16 alone or 1 and 5 in combination (“C01n05”) vs. baseline, for both HIV, ssRNA (Channel 2) and HBV dsDNA (Channel 3) as template, in the presence of three different amplification inhibitors.
  • Figure 9 shows Bar Charts of Mean Delta Ct for the data from Example 1 for compounds 1, 5, 17, 18, 19, 20, and 29 vs. baseline/control (left panel) and for compounds 1, 5, 36, 37, 38, 46, and 53 as well as for positive control Sulfolane vs. baseline/control (right panel) for both HIV, ssRNA (Channel 2) and HBV dsDNA (Channel 3) as template.
  • Figure 10 shows Bar Charts of Mean Delta Ct for the data from Example 2 for compounds 1, 5, 19, 20, and 29 vs. baseline/control (left panel) and for compounds 1, 5, 36, 37, 38, and 53 as well as for positive control Sulfolane vs. baseline/control (right panel) for both HIV, ssRNA (Channel 2) and HBV dsDNA (Channel 3) as template, in the presence of three different amplification inhibitors.
  • kits, and reaction mixtures for amplification of a nucleic acid in the presence of an amplification enhancer is described herein.
  • an amplification enhancer, and optionally polymerase(s), dNTPs (including dATP, dTTP, dCTP, and dGTP), primers, and probes for amplification of a nucleic acid are provided, as are articles of manufacture or kits containing such reagents.
  • the present disclosure is not limited to a specific application of nucleic acid amplification, but will typically be used in applications involving nucleic acid detection.
  • amplifying refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid molecule.
  • Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product.
  • Amplification typically requires the presence of deoxyribonucleoside triphosphates (dNTPs), a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g., MgCh and/or KC1).
  • dNTPs deoxyribonucleoside triphosphates
  • Platinum® Taq a DNA polymerase enzyme
  • an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme e.g., MgCh and/or KC1.
  • hybridizing refers to the annealing of one or more probes to an amplification product.
  • Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.
  • nuclease activity refers to an activity of a nucleic acid polymerase, typically associated with the nucleic acid strand synthesis, whereby nucleotides are removed from the 5’ end of nucleic acid strand.
  • thermostable polymerase refers to a polymerase enzyme that is heat stable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3’ end of each primer and proceeds in the 5’ to 3’ direction along the template strand.
  • Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished, if necessary.
  • nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.
  • nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase that typically adds nucleotides at the 3’ terminal end of a nucleic acid.
  • a nucleotide incorporating biocatalyst such as a polymerase that typically adds nucleotides at the 3’ terminal end of a nucleic acid.
  • nucleic acid sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection.
  • sequence comparison algorithms available to persons of skill or by visual inspection.
  • Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet.
  • modified nucleotide refers to an alteration of nucleotides in which the nucleobase is modified, for example (e.g. to provide a desired property to the nucleotide).
  • exemplary nucleobases that can be used in modified nucleotide include, e.g., a t-butyl benzyl, a C5-methyl-dC, a C5-ethyl- dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a
  • modified nucleotides are known in the art.
  • modified nucleotides are used to modify melting temperatures (T m ) of oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides.
  • T m melting temperatures
  • certain modified nucleotide substitutions can reduce nonspecific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and/or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Patent No. 6,001,611, which is incorporated herein by reference.
  • Other modified nucleotide substitutions may alter the stability of the oligonucleotide, or provide other desirable features.
  • probe refers to synthetically or biologically produced nucleic acids (DNA or RNA), which by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (i.e., preferentially and/or selectively) to “target nucleic acids”.
  • a “probe” can be referred to as a “detection probe” meaning that it detects the target nucleic acid.
  • a probe can comprise one or more modified nucleotides, e.g. to modify the its stability or melting temperature.
  • the described probes can be labeled with at least one fluorescent label.
  • the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher.
  • oligonucleotides to be used as probes can be performed in a manner similar to the design of primers.
  • Embodiments may use a single probe or a pair of probes for detection of the amplification product.
  • the probe(s) used may comprise at least one label and/or at least one quencher moiety.
  • the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequencespecific hybridization to occur but not so long that fidelity is reduced during synthesis.
  • Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.
  • FRET fluorescein
  • a donor fluorescent moiety for example, fluorescein
  • fluorescein is excited at 470 nm by the light source of the LightCycler® Instrument.
  • the fluorescein transfers its energy to an acceptor fluorescent moiety such as LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705).
  • the acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument.
  • the presence of FRET indicates the presence of the target nucleic acid in the sample
  • the absence of FRET indicates the absence of the target nucleic acid in the sample.
  • Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of a test result, however.
  • the nucleic acid can be comprised in a biological sample or non-biological sample, preferably in a biological sample.
  • a biological sample includes, for example, a (human) bodily sample, a microbiological sample, and a cell culture sample.
  • Representative (human) bodily samples that can be used in practicing the methods include, but are not limited to whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissue infections.
  • Non-biological samples include, for example, an environmental sample, such as a soil sample, a water sample and an air sample.
  • the sample can be collected by any method or device designed to adequately hold and store the sample prior to analysis. Such methods and devices are well known in the art.
  • Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that nucleic acids melt at a characteristic temperature called the melting temperature (T m ), which is defined as the temperature at which half of the nucleic acid duplexes have separated into single strands.
  • T m melting temperature
  • the melting temperature of a nucleic acid depends primarily upon its nucleotide composition. Thus, nucleic acid molecules rich in G and C nucleotides have a higher Tm than those having an abundance of A and T nucleotides.
  • the melting temperature of probes can be determined. Similarly, by detecting the temperature at which signal is generated, the annealing temperature of probes can be determined.
  • the melting temperature(s) of the target nucleic acid specific probes from the target amplification products can confirm the presence or absence of target nucleic acid in the sample.
  • Negative control can measure contamination. This ensures that the system and reagents would not give rise to a false positive signal. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequence-specificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.
  • the methods include steps to avoid contamination.
  • an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.
  • Conventional PCR methods in conjunction with FRET technology can be used to practice the methods.
  • a LightCycler® instrument is used. The following patent applications describe real-time PCR as used in the LightCycler® technology: WO 97/46707, WO 97/46714, and WO 97/46712.
  • an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)).
  • a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)
  • fluorescent DNA binding dyes Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength.
  • a double-stranded DNA binding dye such as a nucleic acid intercalating dye also can be used.
  • a melting curve analysis is usually performed for confirmation of the presence of the amplification product.
  • nucleic acid- or signal-amplification methods may also be employed. Examples of such methods include, without limitation, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequencebased amplification (NASBA), self-sustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequencebased amplification
  • SR self-sustained sequence replication
  • SDA strand displacement amplification
  • SMAP 2 smart amplification process version 2
  • Embodiments of the present disclosure further provide for articles of manufacture or kits for amplifying a nucleic acid.
  • An article of manufacture can include primers and probes used to amplify and optionally selectively detect and/or quantitate the target nucleic acid, together with suitable packaging materials, including dNTPs (including dATP, dCTP, dTTP, and dGTP).
  • suitable packaging materials including dNTPs (including dATP, dCTP, dTTP, and dGTP).
  • Representative primers and probes for the amplification and optionally selective detection and/or quantitation of target nucleic acid are capable of hybridizing to target nucleic acid molecules.
  • the kits may also include suitably packaged reagents and materials needed for DNA immobilization, hybridization, and detection, such solid supports, buffers, enzymes, and DNA standards. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that (selectively) amplify and hybridize
  • Articles of manufacture can also include one or more fluorescent moieties for labeling the probes or, alternatively, the probes supplied with the kit can be labeled.
  • an article of manufacture may include a donor and/or an acceptor fluorescent moiety for labeling the target nucleic acid specific probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.
  • Articles of manufacture can also contain a package insert or package label having instructions thereon for using the target nucleic acid specific primers and probes to detect target nucleic acid in a sample.
  • Articles of manufacture may additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerase enzymes, co-factors, or agents to prevent contamination). Such reagents may be specific for one of the commercially available instruments described herein.
  • Embodiments of the present disclosure also provide for a set of primers and one or more detectable probes for the selectively detection and/or quantitation of target nucleic acid in a sample.
  • the polymerase employed was the Z05D polymerase, which is a D580G mutant of the Z05 polymerase, described in, for example, U.S. Patent Nos. US 8,962,293, US 9,102,924, and US 9,738,876.
  • Example 1 One-factorial test of amplification enhancement
  • Table 13 Serial dilution of target panel from PC Stock Table 14: Reaction formulation (One factorial - Taq/ Norro)

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Abstract

La divulgation concerne des procédés d'amplification d'un acide nucléique cible. Les procédés comprennent la mise en oeuvre d'une étape d'amplification comprenant la mise en contact de l'acide nucléique avec une polymérase, des dNTP, un ou plusieurs ensembles d'amorces spécifiques de l'acide nucléique, et un activateur d'amplification qui est le méthylparabène ou un dérivé de celui-ci. L'invention concerne également des kits et des mélanges réactionnels pour l'amplification d'un acide nucléique cible en présence de l'activateur d'amplification, ainsi que des procédés d'amélioration de l'amplification d'acide nucléique.
PCT/EP2025/058616 2024-03-28 2025-03-28 Activateur d'amplification d'acide nucléique Pending WO2025202488A1 (fr)

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