Enhancer of nucleic acid amplification

Abstract

The disclosure relates to methods for amplifying a target nucleic acid. The methods include performing an amplifying step comprising contacting the nucleic acid with a polymerase, dNTPs, one or more set of primers specific for the nucleic acid, and an amplification enhancer that is methyl paraben or a derivative thereof. Also described are kits and reaction mixtures for the amplification of a target nucleic acid in the presence of the amplification enhancer, as well as methods for enhancing nucleic acid amplification.

Description

[0001] ENHANCER OF NUCLEIC ACID AMPLIFICATION

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to enhancers of nucleic acid amplification. The disclosure also relates to methods using the enhancers for amplification, detection, and / or quantitation of a target nucleic acid that may be present in a sample. Further provided are reaction mixtures and kits containing the enhancers.

[0004] BACKGROUND OF THE INVENTION

[0005] Polymerase chain reaction (PCR) is a powerful laboratory technique used to amplify DNA and RNA sequences, allowing researchers to detect and study specific genes or genetic mutations. The process involves repeated cycles of heating and cooling to denature and anneal DNA strands, respectively, and the use of a thermostable DNA polymerase to extend the newly synthesized strands.

[0006] While PCR is a widely used tool in molecular biology, it can be susceptible to inhibitors that interfere with the amplification process. PCR inhibitors can originate from various sources, including the sample itself, such as blood or soil, or from contaminants introduced during DNA extraction or PCR setup. As an example, inhibitors from the different categories can consist of hgDNA, heparin and / or mucin. Inhibitors can reduce PCR efficiency or completely block amplification, resulting in false-negative or inconclusive results. Furthermore, the measurement system capability (repeatability, reproducibility and accuracy) and sensitivity of a PCR based test will be negatively impacted.

[0007] To overcome PCR inhibitors, researchers employ various strategies, including diluting or purifying the sample, modifying reaction conditions, or using additives to reduce inhibition. In some cases, inhibitors can be removed using specialized purification kits or protocols.

[0008] Understanding of potential sources for PCR inhibition and the development of strategies to overcome this is crucial for robust PCR-based research and diagnostic applications. There are various additives acting through different mechanisms that can be used to increase PCR yield or overcome inhibition, including Bovine Serum Albumin (BSA): 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. (1993) Biochemistry 32, 137-44); 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. (1990) 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 (Varadaraj and Skinner, (1994) Gene 140, 1-5); 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). Hence, such known solutions target surfaces of materials or secondary structures of DNA. A stabilization of the DNA amplification reaction as such and a resulting decreased variation, increased sensitivity, and increased robustness vs. interference of the DNA amplification reaction is not described in the art, but shall be analyzed and presented in the following text. Herein, the present disclosure provides novel types of amplification enhancers that can be used in a method for (enhancing) amplification of target nucleic acids. This is particularly useful in circumstances where a (biological) sample may contain PCR inhibitors.

[0009] SUMMARY OF THE INVENTION

[0010] As disclosed herein, it was surprisingly found that adding molecules of the following structure can enhance the efficiency of nucleic acid amplification: wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogem, C / halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogem, C / halogen3or a halogen. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure (i.e., either R3is OH, while R4is a different residue selected from the list for R4(i.e., not OH) or R4is OH, while R3is a different residue selected from the list for R3(i.e., not OH)) and only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2(i.e., if R2is NH2either R3is H, while R4is a different residue selected from the list for R4(i.e., not H) or R4is H, while R3is a different residue selected from the list for R3(i.e., not H)).

[0011] Further, as disclosed herein, it was surprisingly found that adding molecules of the following structure can enhance the efficiency of nucleic acid amplification: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0012] The disclosure thus provides a method for amplifying a nucleic acid, the method comprising (a) contacting the nucleic acid with a polymerase, dNTPs, one or more set of primers specific for the nucleic acid, and an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure (i.e., either R3is OH, while R4is a different residue selected from the list for R4(i.e., not OH) or R4is OH, while R3is a different residue selected from the list for R3(i.e., not OH)) and only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2(i.e., if R2is NH2either R3is H, while R4is a different residue selected from the list for R4(i.e., not H) or R4is H, while R3is a different residue selected from the list for R3(i.e., not H)).

[0013] Further, the disclosure provides a method for amplifying a nucleic acid, the method comprising (a) contacting the nucleic acid with a polymerase, dNTPs, one or more set of primers specific for the nucleic acid, and an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0014] In some embodiments, the method further comprises:

[0015] (b) performing a hybridizing step, wherein the hybridizing step comprises contacting the amplification product from step (a) with one or more probes; and

[0016] (c) performing a detecting step, wherein the detecting step comprises detecting one or more probes hybridized to the nucleic acid.

[0017] In some embodiments, 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).

[0018] The disclosure also provides a method for enhancing amplification of a nucleic acid, the method comprising a step of amplifying the nucleic acid in the presence of an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure (i.e., either R3is OH, while R4is a different residue selected from the list for R4(i.e., not OH) or R4is OH, while R3is a different residue selected from the list for R3(i.e., not OH)) and only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2 (i.e., if R2is NH2 either R3is H, while R4is a different residue selected from the list for R4(i.e., not H) or R4is H, while R3is a different residue selected from the list for R3(i.e., not H)).

[0019] Further, the disclosure also provides a method for enhancing amplification of a nucleic acid, the method comprising a step of amplifying the nucleic acid in the presence of an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0020] In some embodiments, 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.

[0021] In some embodiments, 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. In some embodiments, 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.

[0022] The disclosure also provides a kit for amplifying a nucleic acid, the kit comprising an amplification enhancer as disclosed herein. In some embodiments, the kit for amplifying a nucleic acid comprises:

[0023] (a) one or more polymerases;

[0024] (b) dNTPs;

[0025] (c) one or more set of primers specific for the nucleic acid; and

[0026] (d) an amplification enhancer;

[0027] (e) and optionally one or more set of probes; wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure (i.e., either R3is OH, while R4is a different residue selected from the list for R4(i.e., not OH) or R4is OH, while R3is a different residue selected from the list for R3(i.e., not OH)) and only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2 (i.e., if R2is NH2 either R3is H, while R4is a different residue selected from the list for R4(i.e., not H) or R4is H, while R3is a different residue selected from the list for R3(i.e., not H)).

[0028] Further, the disclosure also provides a kit for amplifying a nucleic acid, wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0029] In some embodiments of the kit, 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.

[0030] The disclosure also provides a reaction mixture for amplifying a nucleic acid, the reaction mixture comprising the amplification enhancer disclosed herein. In some embodiments, the reaction mixture for amplifying a nucleic acid comprises:

[0031] (a) one or more polymerases;

[0032] (b) dNTPs;

[0033] (c) one or more set of primers specific for the nucleic acid;

[0034] (d) an amplification enhancer;

[0035] (e) the nucleic acid

[0036] (f) and optionally one or more set of probes; wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CTL / halogen, CH / halogem, C / halogen3or a halogen; and R4= H, OH, CH3, CTL / halogen, CH / halogem, C / halogen3or a halogen. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure (i.e., either R3is OH, while R4is a different residue selected from the list for R4(i.e., not OH) or R4is OH, while R3is a different residue selected from the list for R3(i.e., not OH)) and only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2 (i.e., if R2is NH2 either R3is H, while R4is a different residue selected from the list for R4(i.e., not H) or R4is H, while R3is a different residue selected from the list for R3(i.e., not H)).

[0037] Further, the disclosure also provides a reaction mixture for amplifying a nucleic acid, wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0038] In some embodiments of the reaction mixture, 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.

[0039] R1can be H, CH3, CH2CH? or CH2CHOHCH2OH. In some embodiments, R1can be H, CH3or CH2CH?. In some embodiments, R1 = H or CH3. In some embodiments, R1 = CH3.

[0040] R2can be OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2. In some embodiments, R2can be OH, O-alkyl, N(alkyl,H)x, NH2; or a halogen, in which x = 1 or 2. In some embodiments, R2can be OH, O-alkyl, N(alkyl,H)xor a halogen, in which x = 1 or 2. The alkyl in the O-alkyl is typically a Ci-C3alkyl, such as a methyl, ethyl, propyl or isopropyl group, preferably methyl or ethyl group, more preferably a methyl group (CH3). When R2= O-alkyl, R1is preferably CH3or CH2CH?, more preferably CH3. The alkyl in N(alkyl,H)x is typically a Ci- C3alkyl, such as a methyl, ethyl, propyl or isopropyl group, preferably methyl (CH3) or ethyl (CH2CH3) group, more preferably a methyl group (CH3). In some embodiments, N(alkyl,H)x is NH(alkyl). If R2is NH2 only either one of R3and R4may be H in the same amplification enhancer structure. When R2is NH2, R3is typically not H (in such embodiments, R3can be OH or CH3, for example). In some embodiments, R2can be OH, O-alkyl or N(alkyl,H)x. In some embodiments, R2= OH, NHCH3 or a halogen. In some embodiments, R2= OH or NHCH3. In some embodiments, R2= OH.

[0041] R3can be H, OH, CH3, CH2 / halogen, CH / halogem, C / halogem or a halogen. CH2 / halogen, CH / halogen2 or C / halogem represent halogen- substituted methyl groups. In some embodiments, R3= H, OH or CH3. In some embodiments, R3= H or OH. In some embodiments, R3= OH or CH3. In some embodiments, R3= OH.

[0042] R4can be H, OH, CH3, CH2 / halogen, CH / halogem, C / halogem or a halogen. CH2 / halogen, CH / halogen2 or C / halogem represent halogen- substituted methyl groups. In some embodiments, R4= H, OH or CH3. In some embodiments, R4= H or OH. In some embodiments, R4= H. Herein, only either one of R3and R4may be OH in the same amplification enhancer structure. The halogen can be selected from, for example, F, Cl and Br. Preferably, the halogen is selected from F and Br. When more than one halogen is present in the amplification enhancer, the halogens can be independently selected from, for example, F, Cl and Br, preferably from F and Br. When more than one halogen is present in the amplification enhancer, the halogens can also be of the same type. This will typically be the case of a halogen-substituted methyl group. For example, CH2 / halogen, CH / halogem or C / halogem can be CH2F, CHF2 or CF3, respectively, and preferably CF3.

[0043] In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, and R3= H, OH or CH3. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl), and R3= H, OH or CH3. In some embodiments, R1= H or CH3, R2= OH, and R3= H, OH or CH3. In some embodiments, R1= CH3, R2= OH, and R3= H, OH or CH3. In some embodiments, R1= CH3, R2= OH, and R3= H or OH. In some embodiments, R1= H, R2= OH, and R3= H, OH, F or CF3.

[0044] In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, R3= H, OH or CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl), R3= H, OH or CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH, R3= H, OH or CH3 and R4=H. In some embodiments, R1= CH3, R2= OH, R3= H, OH or CH3 and R4=H. In some embodiments, R1= CH3, R2= OH, R3= H or OH and R4=H. In some embodiments, R1= H, R2= OH, R3= H, OH, F or CF3 and R4=H. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl), NH2 or a halogen and R3= OH, CH3, CF3 or a halogen. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, and R3= OH, CH3, CF3 or a halogen. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl) and R3= OH, CH3, CF3 or a halogen. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, and R3= OH or CH3. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, and R3= OH, F, CH3. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl), and R3= OH or CH3. In some embodiments, R1= H or CH3, R2= OH, and R3= OH or CH3. In some embodiments, R1= CH3, R2= OH, and R3= OH or CH3. In some embodiments, R1= CH3, R2= OH, and R3 = OH.

[0045] In some embodiments, R1= H or CH3, R2= OH, NH(alkyl), NH2 or a halogen, R3= OH, CH3, CF3 or a halogen and R4=H. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, R3= OH, CH3, CF3 or a halogen and R4=H. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl), R3= OH, CH3, CF3 or a halogen and R4=H. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, R3= OH, F, CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH, NH(alkyl) or a halogen, R3= OH or CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH or NH(alkyl), R3= OH or CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH, R3= OH or CH3 and R4=H. In some embodiments, R1= H or CH3, R2= OH, R3= OH or CH3 and R4=H. In some embodiments, R1= CH3, R2= OH, R3= OH or CH3 and R4=H. In some embodiments, R1= CH3, R2= OH, R3 = OH and R4=H.

[0046] In some embodiments, 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, ethylparaben, 4-Hydroxy-N,N-dimethylbenzamide, 4-Acetamidophenol, and thymol. In some embodiments, 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. In some embodiments, the amplification enhancer is selected 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, ethylparaben, and thymol. In some embodiments, the amplification enhancer is selected 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-m ethylbenzoate, and ethylparaben. In some embodiments, the amplification enhancer is selected from methylparaben, methyl-2,4-dihydroxy benzoate, Methyl 4-hydroxy-2-methylbenzoate, ethylparaben and thymol. In some embodiments, the amplification enhancer is selected from methylparaben, 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. In some embodiments, the amplification enhancer is selected 4-Hydroxy-N,N- dimethylbenzamide, 4- Acetamidophenol, and thymol. In some embodiments, the amplification enhancer is thymol.

[0047] In some embodiments, the amplification enhancer is not methylparaben. Thus, in some embodiments, 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. In some embodiments, 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. In some embodiments, the amplification enhancer is selected from 4-hydroxybenzoic acid, methyl-2,4-dihydroxy- benzoate, 4-hydroxy-2-methylbenzoic acid, 4-(methylamine)benzoic acid, 4-amino-salicylic acid, Arm-43, Methyl 4-hydroxy-2-m ethylbenzoate, ethylparaben, and thymol. In some embodiments, the amplification enhancer is selected from 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-m ethylbenzoate, and ethylparaben. In some embodiments, the amplification enhancer is selected from 4-hydroxybenzoic acid, methyl-2,4- dihydroxybenzoate, 4-hydroxy-2-methylbenzoic acid and 4-(methylamine)benzoic acid. In some embodiments, 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.

[0048] In some embodiments, the number of carbon atoms comprised in the amplification enhancer is 30 or less, 25 or less, 23 or less, 20 or less, 15 or less, such as 14 or less, 13 or less, 12 or less, 11 or less or 10 or less.

[0049] It is also possible to use two or more enhancers in combination. In some embodiments, 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. In some embodiments, 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. In some embodiments, the amplification enhancer is a mixture of two or more enhancers selected from consisting of 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, ethylparaben, and thymol. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0050] When the amplification enhancer is a mixture of two or more enhancers, 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).

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0052] In the structures shown herein, when not all natural valences of an atom are filled by named groups, it should be understood that the unfilled valences are filled by hydrogen. When a wavy line in a structure intersects a bond, then the intersected bond is the location where the structure joins to the remainder of a molecule.

[0053] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.

[0054] Exemplary embodiments of the disclosure are as described in the following items:

[0055] [1] A method for amplifying a nucleic acid, the method comprising:

[0056] (a) contacting the nucleic acid with a polymerase, dNTPs, one or more set of primers specific for the nucleic acid, and an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2; or wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0057] [2] The method of item 1, further comprising:

[0058] (b) performing a hybridizing step, wherein the hybridizing step comprises contacting the amplification product from step (a) with one or more probes; and

[0059] (c) performing a detecting step, wherein the detecting step comprises detecting one or more probes hybridized to the nucleic acid.

[0060] [3] The method of item 1 or 2, wherein the polymerase is a DNA polymerase.

[0061] [4] The method of any one of the preceding items, wherein the dNTPs comprise dATP, dTTP, dCTP, and dGTP.

[0062] [5] The method of any one of the preceding items, wherein the nucleic acid is an RNA or a DNA.

[0063] [6] The method of any one of the preceding items, wherein the nucleic acid is comprised in a biological sample.

[0064] [7] The method of item 6, wherein the biological sample is a bodily sample, such as blood, plasma, or urine.

[0065] [8] The method of any one of the preceding items, wherein the presence of the amplification enhancer results in a faster amplification of the nucleic acid compared to the amplification under the same conditions but without the amplification enhancer.

[0066] [9] The method of any one of the preceding items, wherein R1 = H or CH3, preferably CH3.

[0067]

[0010] The method of any one of the preceding items, wherein R2 = OH or NHCH3, preferably OH.

[0011] The method of any one of the preceding items, wherein R3= H, OH or CH3, preferably OH or CH3.

[0068]

[0012] The method of any one of the preceding items, wherein R4= H, OH or CH3, preferably H.

[0069]

[0013] The method of any one of the preceding items, 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.

[0070]

[0014] The method of any one of the preceding items, wherein the amplification enhancer is methyl-2,4-dihydroxy benzoate.

[0071]

[0015] The method of any one of the preceding items, wherein the amplification enhancer is a mixture of methylparaben and methyl-2,4-dihydroxy benzoate.

[0072]

[0016] 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).

[0073]

[0017] A kit for amplifying a nucleic acid, the kit comprising:

[0074] (a) one or more polymerases;

[0075] (b) dNTPs;

[0076] (c) one or more set of primers specific for the nucleic acid; and

[0077] (d) an amplification enhancer;

[0078] (e) and optionally one or more set of probes; wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2; or wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0079]

[0018] The kit of item 17, wherein the polymerase is a DNA polymerase.

[0080]

[0019] The kit of item 17 or 18, wherein the dNTPs comprise dATP, dTTP, dCTP, and dGTP.

[0081]

[0020] The kit of any one of items 17-19, wherein the nucleic acid is an RNA or a DNA.

[0082]

[0021] The kit of any one of items 17-20, wherein the kit is for amplifying a nucleic acid comprised in a biological sample.

[0083]

[0022] The kit of item 21, wherein the biological sample is a bodily sample, such as blood, plasma, or urine.

[0084]

[0023] The kit of any one of items 17-22, wherein R1= H or CH3, preferably CH3.

[0085]

[0024] The kit of any one of items 17-23, wherein R2= OH or NHCH3, preferably OH.

[0086]

[0025] The kit of any one of items 17-24, wherein R3= H, OH or CH3, preferably OH or CH3.

[0087]

[0026] The kit of any one of items 17-25, wherein R4= H, OH or CH3, preferably H.

[0088]

[0027] The kit 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.

[0089]

[0028] The kit of any one of items 17-27, wherein the amplification enhancer is methyl-2,4- dihydroxy benzoate.

[0090]

[0029] The kit of any one of items 17-28, wherein the amplification enhancer is a mixture of methylparaben and methyl-2,4-dihydroxy benzoate.

[0091]

[0030] The kit of any one of items 17-29, wherein the amplification enhancer is comprised in a composition configured to provide a final concentration of the amplification enhancer of 0.01% to 0.09% (m / V) during the amplification of the nucleic acid.

[0092]

[0031] A method for enhancing amplification of a nucleic acid, the method comprising a step of amplifying the nucleic acid in the presence of an amplification enhancer, wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure ifR2is NH2; or wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0093]

[0032] The method of item 31, wherein 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.

[0094]

[0033] The method of item 32, wherein the polymerase is a DNA polymerase.

[0095]

[0034] The method of item 32 or 33, wherein the dNTPs comprise dATP, dTTP, dCTP, and dGTP.

[0096]

[0035] The method of any one of items 31-34, wherein the nucleic acid is an RNA or a DNA.

[0097]

[0036] The method of any one of items 31-35, wherein the nucleic acid is comprised in a biological sample.

[0098]

[0037] The method of item 36, wherein the biological sample is a bodily sample, such as blood, plasma, or urine.

[0099]

[0038] The method of any one of items 31-37, wherein the presence of the amplification enhancer results in a faster amplification of the nucleic acid compared to the amplification under the same conditions but without the amplification enhancer.

[0100]

[0039] The method of any one of items 31-38, wherein R1= H or CH3, preferably CH3.

[0101]

[0040] The method of any one of items 31-39, wherein R2= OH or NHCH3, preferably OH.

[0102]

[0041] The method of any one of items 31-40, wherein R3= H, OH or CH3, preferably OH or CH3.

[0103]

[0042] The method of any one of items 31-41, wherein R4= H, OH or CH3, preferably H.

[0104]

[0043] The method of any one of items 31-42, 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.

[0105]

[0044] The method of any one of items 31-43, wherein the amplification enhancer is methyl-2,4- dihydroxy benzoate.

[0045] The method of any one of items 31-44, wherein the amplification enhancer is a mixture of methylparaben and methyl-2,4-dihydroxy benzoate.

[0106]

[0046] The method of any one of items 31-45, wherein the amplification enhancer is present at a concentration of 0.01% to 0.09% (m / V) in the step of amplifying the nucleic acid.

[0107]

[0047] A reaction mixture for amplifying a nucleic acid, the reaction mixture comprising:

[0108] (a) one or more polymerases;

[0109] (b) dNTPs;

[0110] (c) one or more set of primers specific for the nucleic acid;

[0111] (d) an amplification enhancer;

[0112] (e) the nucleic acid

[0113] (f) and optionally one or more set of probes; wherein the amplification enhancer has the following structure: wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CTL / halogen, CH / halogem, C7halogen3or a halogen; and R4= H, OH, CH3, CTb / halogen, CH / halogem, C7halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure ifR2is NH2; or wherein the amplification enhancer has the following structure: wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

[0114]

[0048] The reaction mixture of item 47, wherein the polymerase is a DNA polymerase.

[0115]

[0049] The reaction mixture of item 47 or 48, wherein the dNTPs comprise dATP, dTTP, dCTP, and dGTP.

[0116]

[0050] The reaction mixture of any one of items 47-49, wherein the nucleic acid is an RNA or a DNA.

[0051] 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.

[0117]

[0052] The reaction mixture of item 51, wherein the biological sample is a bodily sample, such as blood, plasma, or urine.

[0118]

[0053] The reaction mixture of any one of items 47-52, wherein R1= H or CH3, preferably CH3.

[0119]

[0054] The reaction mixture of any one of items 47-53, wherein R2= OH or NHCH3, preferably OH.

[0120]

[0055] The reaction mixture of any one of items 47-54, wherein R3= H, OH or CH3, preferably OH or CH3.

[0121]

[0056] The reaction mixture of any one of items 47-55, wherein R4= H, OH or CH3, preferably H.

[0122]

[0057] The 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.

[0123]

[0058] The reaction mixture of any one of items 47-57, wherein the amplification enhancer is methyl-2,4-dihydroxy benzoate.

[0124]

[0059] The reaction mixture of any one of items 47-58, wherein the amplification enhancer is a mixture of methylparaben and methyl-2,4-dihydroxy benzoate.

[0125]

[0060] The reaction mixture of any one of items 47-59, wherein the amplification enhancer is present at a concentration of 0.01% to 0.09% (m / V).

[0126] BRIEF DESCRIPTION OF THE FIGURES

[0127] Figure 1 shows an overview over the experimental design for example 1 and example 2.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] Figure 5 shows the experimental design for a one-factorial test using Taq Polymerase for Norro GG2 ssRNA as template.

[0132] 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

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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. DETAILED DESCRIPTION OF THE INVENTION

[0137] Methods, kits, and reaction mixtures for amplification of a nucleic acid in the presence of an amplification enhancer is described herein. In particular, 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.

[0138] As used herein, the term “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).

[0139] The term “primer” as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily to oligonucleotides but also to modified oligonucleotides (comprising one or more modified nucleotides) that are able to “prime” DNA synthesis by a template-dependent DNA polymerase, i.e., the 3’-end of the, e.g., oligonucleotide provides a free 3’-OH group where further "nucleotides" may be attached by a template-dependent DNA polymerase establishing 3’ to 5’ phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.

[0140] The term “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.

[0141] The term “5’ to 3’ 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.

[0142] The term “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.

[0143] The term “complement thereof’ refers to nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.

[0144] The term “extension” or “elongation” when used with respect to nucleic acids refers to when additional nucleotides (or other analogous molecules) are incorporated into the nucleic acids. For example, a 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.

[0145] The terms “identical” or percent “identity” in the context of two or more 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. 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. 3 :266-272, Madden et al. (1996) “Applications of network BLAST server” Meth. Enzymol. 266: 131-141, Altschul et al. (1997) “Gapped BLAST and PSLBLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation” Genome Res. 7:649-656, which are each incorporated herein by reference.

[0146] A “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 C7-propargylamino-dG, a 7-deaza-2-deoxy -xanthosine, a pyrazolo- pyrimidine analog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2’-0-methyl ribo-U, 2’-0-methyl ribo-C, an N4-ethyl-dC, an N6-methyl-dA, a 5-propynyl dU, a 5-propynyl dC, 7-deaza- deoxyguanosine (deaza G (u-deaza)) and the like. Many other modified nucleotides are known in the art. In certain embodiments, modified nucleotides are used to modify melting temperatures (Tm) of oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides. To further illustrate, 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.

[0147] The term “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.

[0148] In some embodiments, the described probes can be labeled with at least one fluorescent label. In one embodiment, the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher.

[0149] Designing 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. Depending on the embodiment, the probe(s) used may comprise at least one label and / or at least one quencher moiety. As with the primers, 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.

[0150] Polymerase-mediated nucleic acid amplification

[0151] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional polymerase-mediated nucleic acid amplification techniques. Polymerase-mediated amplification typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides capable of acting as points of initiation of nucleic acid synthesis within target nucleic acid sequences. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating.

[0152] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in polymerase-mediated amplification. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90 °C to about 105 °C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5 min).

[0153] If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence. The temperature for annealing is usually from about 35 °C to about 65 °C (e.g., about 40 °C to about 60 °C; about 45 °C to about 50 °C). Annealing times can be from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40 °C to about 80 °C (e.g., about 50 °C to about 70 °C; about 60 °C). Extension times can be from about 10 sec to about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30 sec to about 2 min).

[0154] Polymerase-mediated amplification can employ nucleic acid such as RNA (including e.g. polyA RNA) or DNA (including e.g. genomic DNA and cDNA). The template nucleic acid needs not be purified; it may be a minor fraction of a complex mixture, such as target RNA or DNA contained in human cells. Target nucleic acid molecules may be extracted from a biological sample by routine techniques such as those described in Diagnostic Molecular Microbiology. Principles and Applications (Persing el al. (eds), 1993, American Society for Microbiology, Washington D.C.). Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0155] The oligonucleotide primers are combined with amplification reagents under reaction conditions that induce primer extension. Such amplification reagents include, but are not limited to, one or more polymerases, and dNTPs (including dATP, dTTP, dCTP, and dGTP). For example, chain extension reactions generally include 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 15 mM MgCh, 0.001% (w / v) gelatin, 0.5-1.0 pg denatured template nucleic acid (such as DNA), 50 pmoles of each oligonucleotide primer, 2.5 U of polymerase, and 10% DMSO). The reactions usually contain 150 to 320 pM each of dATP, dCTP, dTTP, and dGTP, or one or more modified versions thereof.

[0156] The newly-synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target nucleic acid molecules. This is typically called a polymerase-chain reaction (PCR). The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (z.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.

[0157] Detection and / or Quantitation of the nucleic acid Amplified Product (Amplicon)

[0158] The present disclosure provides methods for selectively detecting and / or quantitating a nucleic acid in a biological or non-biological sample. The methods include performing at least one cycling step that includes amplifying a portion of a target nucleic acid from a sample using one or more pairs of target specific primers, and a FRET detecting step. Multiple cycling steps can be performed, preferably in a thermocycler. Methods can be performed using the target specific primers and probes to selectively detect and / or quantitate the target nucleic acid, wherein the presence of target nucleic acid, and the detection of target nucleic acid indicates the presence of target nucleic acid in the sample.

[0159] As described herein, amplification products can be detected using labeled hybridization probes that take advantage of FRET technology. FRET technology (see, for example, U.S. Patent Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on a concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and / or quantitated. The donor typically transfers the energy to the acceptor when the donor is excited by light radiation with a suitable wavelength. The acceptor typically re-emits the transferred energy in the form of light radiation with a different wavelength. In certain systems, non-fluorescent energy can be transferred between donor and acceptor moieties, by way of biomolecules that include substantially non-fluorescent donor moieties (see, for example, US Patent. No. 7,741,467).

[0160] One FRET format utilizes TaqMan® technology to detect the presence or absence of an amplification product, and hence, the presence or absence of target nucleic acid. TaqMan® technology utilizes one single-stranded hybridization probe labeled with, e.g., one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety or a dark quencher according to the principles of FRET. The second moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (z.e., the amplification product) and is degraded by the 5’ to 3’ nuclease activity of, e.g., the Taq Polymerase during the subsequent elongation phase. As a result, the fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected. By way of example, an ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology, and is suitable for performing the methods described herein for selectively detecting and / or quantitating target nucleic acid in the sample.

[0161] Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the target nucleic acids (i.e., amplification products), the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

[0162] Another common format of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and are generally designed to hybridize in close proximity to each other in a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, for example, fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, 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. Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules. If amplification of target nucleic acid occurs and an amplification product is produced, the step of hybridizing results in a detectable signal based upon FRET between the members of the pair of probes.

[0163] Generally, the presence of FRET indicates the presence of the target nucleic acid in the sample, and 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.

[0164] 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.

[0165] 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.

[0166] 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 (Tm), which is defined as the temperature at which half of the nucleic acid duplexes have separated into single strands. 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. By detecting the temperature at which signal is lost, 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.

[0167] Within each thermocycler run, control samples can be cycled as well. Positive control samples can amplify target nucleic acid control template (other than described amplification products of target genes) using, for example, control primers and control probes. Positive control samples can also amplify, for example, a plasmid construct containing the target nucleic acid molecules. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run side-by-side with the patients' samples using the same primers and probe as used for detection of the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include a negative control that, for example, lacks target template DNA. 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.

[0168] In an embodiment, the methods include steps to avoid contamination. For example, 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. In one embodiment, 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.

[0169] The LightCycler® can be operated using a PC workstation and can utilize a Windows NT operating system. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit. The software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.

[0170] As an alternative to FRET, 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)). 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. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product.

[0171] One of skill in the art would appreciate that other 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).

[0172] It is understood that the embodiments of the present disclosure are not limited by the configuration of one or more commercially available instruments.

[0173] Articles of Manufacture / Kits

[0174] 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). 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. In addition, 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 to nucleic acid target molecules are provided.

[0175] 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. For example, 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.

[0176] 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.

[0177] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0178] EXAMPLES

[0179] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. In some of the following Examples, 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.

[0180] Example 1 : One-factorial test of amplification enhancement

[0181] It was surprisingly found out that methylparaben has an enhancing effect on nucleic acid amplification, and then further tested a series of analogous compounds for such an enhancing effect. An overview over the experimental design for example 1 and example 2 is shown in Figure

[0182] 1.

[0183] Assays were conducted with either ssRNA (from HIV) or dsDNA (from HBV) as template nucleic acid. The experimental setup for the One factorial screening - Z05D / MPX was as follows:

[0184] 1. Provide 11.1% (m / V) enhancer in 2: 1 (V / V) EtOH:DMSO and dilute in Mastermix (MMX) R2 by factor 37, clarify if partially precipitation occurred

[0185] 2. Provide target panels in lOmM Tris pH8 (Tables 2 and 3)

[0186] 3. Assemble reaction according to formulation (Table 1)

[0187] 4. Amplify and detect according to PCR / fluorescence profile (Tables 8 and 9)

[0188] 5. For reagents and functional components (Tables 10-12) Table 1: Reaction formulation (One factorial - Z05D / MPX)

[0189] *8, 16, 24xLOD (limit of detection) final concentration in reaction (Consider concentration factor 13 of sample preparation)

[0190] Table 2: Arithmetic dilution of target panels from lOOOxLOD

[0191] Table 3: LOD MPX Assay

[0192] Results from one-factorial screening tests are shown in Figure 2.

[0193] Example 2: Two-factorial test of amplification enhancement in presence of known inhibitors

[0194] It was next tested if the compounds could also enhance amplification in the presence of known amplification inhibitors; in other words, if the compounds could bypass the inhibition mediated from the inhibitors. An overview over the experimental design for example 2 is shown in Figure 1. The experimental Setup for the Two factorial screening - Z05D / MPX was as follows:

[0195] 1. Provide 11.1% (m / V) Enhancer in 2: 1 (V / V) EtOELDMSO and dilute in MMX R2 by factor 37, clarify if partially precipitation occurred

[0196] 2. Provide Inhibitor panel in lOmM Tris pH8 (Table 5)

[0197] 3. Provide target panel in lOmM Tris pH8 (Tables 6 and 7)

[0198] 4. Assemble reaction according to formulation (Table 4)

[0199] 5. Amplify and detect according to PCR / fluorescence Profile (Tables 8 and 9)

[0200] 6. For reagents and functional components (Tables 10-12) Table 4: Reaction formulation (Two factorial - Z05D / MPX)

[0201] *8xLOD final concentration in reaction (Consider concentration factor 13 of sample preparation)

[0202] Table 5: Preparation Inhibitor Panels

[0203] **inhibition tolerance of assay was determined preliminary, input limits can vary especially for hgDNA (Lot to Lot variation)

[0204] Table 6: Arithmetic dilution of target panel from XOOOxLOD

[0205] Table 7: LOD MPX Assay Table 8: PCR Profile Table 9: Detection - Fluorescence Read Out

[0206] Materials used in the examples was as follows:

[0207] Table 10: Reagents

[0208] Table 11: Enhancer List

[0209] Table 12: Inhibitor List

[0210] Results from two-factorial tests are shown in Figure 3. Further results from two-factorial tests for top three candidates are shown in Figure 4.

[0211] The preceding data were raised using Z05D polymerase. Further tests were conducted using Taq polymerase as follows. Experimental design is shown in Figure 5 and the results for the top three candidates are shown in Figure 6. The experimental setup for the One factorial screening - Taq / Norro was as follows: 1. Provide 11.1% (m / V) Enhancer in 2: 1 (V / V) EtOELDMSO and dilute in Roche Master by factor 32, clarify if partially precipitation occurred

[0212] 2. Prep of PSR: Add 50uL 30mM Tris pH 8 (tube with yellow cap), vortex and spin down

[0213] 3. Prep of PC: Add 160uL 30mM Tris buffer pH 8 (tube with black cap - PC), mix by pipetting up and down 10 times and spin down 4. Provide target panels in 30mM Tris pH8 (Table 13)

[0214] 5. Assemble reaction according to formulation (Table 14)

[0215] 6. Amplify and detect according to PCR / fluorescence Profile (Tables 8 and 9)

[0216] Table 13: Serial dilution of target panel from PC Stock Table 14: Reaction formulation (One factorial - Taq / Norro)

[0217] Summary

[0218] The data from Example 1 and 2 for the five top candidate compounds 1, 3, 5, 15 and 16 as well as the combination of compounds 1 and 5 is summarized in Figure 7 and Figure 8. Additional data for other high performing candidate compounds 17, 18, 19, 20, 29, 36, and 37 as well as 38, 46 and 53 is summarized in Figure 9 and Figure 10.

[0219] The results for compounds tested are summarized in Table 15 below, showing that compounds 1, 3, 5, 7, 9, 11, 15, 16-20, 29, 36-38, 44, 46 and 53 tested had enhanced amplification efficiency as evidenced by a negative shift in Ct values in the presence of the compounds. Of these, compounds 1, 3, 5, 15, 16, 18, 29, 36, 37, 38, 46 and 53 exhibited a strong enhancing effect, while compounds 7, 9, 11, 17, 19, 20, and 44 exhibited a moderate enhancing effect.

[0220] Moreover, compounds 1, 5, 15, 16, 19, 20, 29, 36-38 and 53 also had an inhibitor bypass effect, i.e. these compounds enhanced amplification efficiency even in the presence of known PCR inhibitors. The combination of compounds 1, 5, 29, 36-38 and 53 worked particularly well, both in terms of amplification enhancement and inhibitor bypassing.

[0221] The data allowed formulating a general structural formula of compounds that achieve an amplification enhancing effect and / or an inhibitor bypass effect, as reflected in the general structural formula disclosed herein.

[0222] Table 15: Results for exemplary compounds

[0223] Example 3 : Control setup for one- and two factorial test of amplification enhancement

[0224] In order to validate the efficiency of the chosen method and to distinguish enhancing effects from normal baseline variation a control reaction was performed using literature-known strong PCR enhancing moieties, such as sulfolane (Chakrabarti, et al. Biotechniques (2002) 32(4):866, 868, 870-2, 874.).

[0225] Table 16: Sulfolane

[0226] Herein, sulfolane was employed to test the chosen read-out parameter, "delta CT values" (CT value obtained with sulfolane - CT value from Baseline) with the selected PCR setup of Examples 1 and 2. This value correlated well with the expected enhancing effect for both 1 -factorial and 2- factorial experimental setups showing a mean delta CT of at least 1 for ssRNA and of at least 0.6 for dsDNA in both 1 -factorial and 2-factorial testing as shown in Figures 9 and 10.

[0227] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes.

Claims

CLAIMS1. A method for amplifying a nucleic acid, the method comprising(a) contacting the nucleic acid with a polymerase, dNTPs, one or more set of primers specific for the nucleic acid, and an amplification enhancer, wherein the amplification enhancer has the following structure:wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C7halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure if R2is NH2; or wherein the amplification enhancer has the following structure:wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

2. The method of claim 1, wherein the presence of the amplification enhancer results in a faster amplification of the nucleic acid compared to the amplification under the same conditions but without the amplification enhancer.

3. The method of claim 1 or 2, wherein a) R1= H or CH3, preferably CH3, and / or b) R2= OH or NHCH3, preferably OH, and / orc) R3= H, OH or CH3, preferably OH or CH3, and / or d) R4= H, OH or CH3, preferably H.

4. The method of any one of the preceding claims, 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.

5. The method of any one of the preceding claims, wherein the amplification enhancer is present in step (a) at a concentration of 0.01% to 0.09% (m / V).

6. A kit for amplifying a nucleic acid, the kit comprising:(a) one or more polymerases;(b) dNTPs;(c) one or more set of primers specific for the nucleic acid; and(d) an amplification enhancer;(e) and optionally one or more set of probes; wherein the amplification enhancer has the following structure:wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CTL / halogen, CH / halogem, C / halogen3 or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogem, C / halogem or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure ifR2is NH2; or wherein the amplification enhancer has the following structure:wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

7. The kit of claim 6, wherein a) R1= H or CH3, preferably CH3, and / or b) R2= OH or NHCH3, preferably OH, and / or c) R3= H, OH or CH3, preferably OH or CH3, and / or d) R4= H, OH or CH3, preferably H.

8. The kit of claim 6 or 7, wherein the amplification enhancer is methyl-2,4-dihydroxy benzoate and / or methylparaben, and / or wherein the amplification enhancer is comprised in a composition configured to provide a final concentration of the amplification enhancer of 0.01% to 0.09% (m / V) during the amplification of the nucleic acid.

9. A method for enhancing amplification of a nucleic acid, the method comprising a step of amplifying the nucleic acid in the presence of an amplification enhancer, wherein the amplification enhancer has the following structure:wherein R1= CH3, H, CH2CH3or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / halogen2, C / halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; and wherein only either one of R3and R4may be H in the same amplification enhancer structure ifR2is NH2; or wherein the amplification enhancer has the following structure:wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

10. The method of claim 9, wherein the presence of the amplification enhancer results in a faster amplification of the nucleic acid compared to the amplification under the same conditions but without the amplification enhancer.

11. The method of claim 9 or 10, wherein a) R1= H or CH3, preferably CH3, and / or b) R2= OH or NHCH3, preferably OH, and / or c) R3= H, OH or CH3, preferably OH or CH3, and / or d) R4= H, OH or CH3, preferably H.

12. The method of any one of claims 9-11, wherein the amplification enhancer is methyl-2,4- dihydroxy benzoate and / or methylparaben, and / or wherein the amplification enhancer is present at a concentration of 0.01% to 0.09% (m / V) in the step of amplifying the nucleic acid.

13. A reaction mixture for amplifying a nucleic acid, the reaction mixture comprising:(a) one or more polymerases;(b) dNTPs;(c) one or more set of primers specific for the nucleic acid;(d) an amplification enhancer;(e) the nucleic acid(f) and optionally one or more set of probes; wherein the amplification enhancer has the following structure:wherein R1= CH3, H, CH2CH3 or CH2CHOHCH2OH; R2= OH, O-alkyl, N(alkyl,H)x, NH2; N3 or a halogen, in which x = 1 or 2; R3= OH, H, CH3, CH2 / halogen, CH / halogen2, C / halogens or a halogen; and R4= H, OH, CH3, CH2 / halogen, CH / haloge , C7halogen3or a halogen, wherein only either one of R3and R4may be OH in the same amplification enhancer structure; andwherein only either one of R3and R4may be H in the same amplification enhancer structure ifR2is NH2; or wherein the amplification enhancer has the following structure:wherein R5= NH-C(O)-CH3, C(O)-N(CH3)2or CH(CH3)2; R6= H, OH or a halogen; R7= OH or CH3.

14. The reaction mixture of claim 13, wherein a) R1= H or CH3, preferably CH3, and / or b) R2= OH or NHCH3, preferably OH, and / or c) R3= H, OH or CH3, preferably OH or CH3, and / or d) R4= H, OH or CH3, preferably H.

15. The reaction mixture of claims 13 or 14, wherein the amplification enhancer is methyl-2,4- dihydroxy benzoate and / or methylparaben, and / or wherein the amplification enhancer is present at a concentration of 0.01% to 0.09% (m / V).

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