Composition for reducing inhibition of nucleic acid amplification
A composition for reducing the inhibitory effects of contaminants on nucleic acid amplification is provided. The composition includes a plurality of zirconium oxide particles, a non-ionic surfactant, and an organic iron-chelating reagent. The organic iron-chelating reagent has a first affinity constant greater than or equal to 10 4.2 with respect to ferric iron and a second affinity constant less than 10 3.8 with respect to magnesium, wherein the first affinity constant and the second affinity constant are determined in deionized water at pH 8.45 and 20° C. Optionally, the composition includes polyvinylpyrrolidone. Optionally, the composition comprises water. The composition has a pH of about 8.45 to 8.85. Methods of using the composition to prepare a sample for nucleic acid amplification are also provided.
1. An aqueous composition for eliminating sample inhibition in a nucleic acid amplification reaction, said aqueous composition comprising:
a plurality of zirconium oxide particles;
a non-ionic surfactant at a concentration greater than or equal to 0.005% (mass/volume); and
an organic iron-chelating reagent;
wherein the composition has a pH of about 8.45 to 8.85;
wherein the organic iron-chelating reagent has a first affinity constant greater than or equal to 10 4.2 with respect to ferric iron and a second affinity constant less than 10 3.8 with respect to magnesium, wherein the first affinity constant and the second affinity constant are determined in 20° C. deionized water at pH 8.45;
and wherein the composition further comprises a nanoparticle dispersion stabilizer, polyvinylpyrrolidone, or both a nanoparticle dispersion stabilizer and polyvinylpyrrolidone.
2. The aqueous composition of claim 1 , wherein the plurality of particles has a mean particle size that is less than or equal to 100 nm.
3. The aqueous composition of claim 1 , wherein the organic iron-chelating reagent comprises a plurality of carboxylate groups.
4. The aqueous composition of claim 1 , further comprising ferric iron.
5. The aqueous composition of claim 1 , further comprising a nanoparticle dispersion stabilizer.
6. The aqueous composition of claim 1 , wherein the non-ionic surfactant has a Hydrophilic-lipophilic balance of about 11 to about 16.
7. The aqueous composition of claim 1 , further comprising polyvinylpyrrolidone.
8. The aqueous composition of claim 1 , further comprising an indicator dye.
9. A nucleic acid amplification method, said method comprising:
a) contacting a composition of claim 1 ;
b) subjecting the aqueous mixture of step a) to a thermal lysis process; and
c) after step b), subjecting a portion of the aqueous mixture to a nucleic acid amplification process.
10. The method of claim 9 , wherein the sample is incubated in a culture broth prior to step a).
11. The method of claim 9 , wherein the composition further comprises a buffering agent, an enhancer for facilitating a LAMP-BART nucleic acid amplification reaction or an enhancer for facilitating a qPCR reaction.
12. The method of claim 9 , wherein subjecting the mixture to thermal lysis comprises heating the mixture to about 100° C. for about 15 minutes.
13. A kit, comprising:
a plurality of zirconium oxide particles;
a non-ionic surfactant; and
an organic iron-chelating reagent;
wherein the organic iron-chelating reagent has a first affinity constant greater than or equal to 10 4.2 with respect to ferric iron and a second affinity constant less than 10 3.8 with respect to magnesium, wherein the first affinity constant and the second affinity constant are determined in 20° C. deionized water at pH 8.45; and
a nanoparticle dispersion stabilizer, polyvinylpyrrolidone, or both a nanoparticle dispersion stabilizer and polyvinylpyrrolidone.
14. The kit of claim 13 , wherein the organic iron-chelating reagent comprises a plurality of carboxylate groups.
15. The kit of claim 13 , further comprising ferric iron.
16. The kit of claim 13 , further comprising a nanoparticle dispersion stabilizer.
17. The kit of claim 13 , wherein the non-ionic surfactant has a Hydrophilic-lipophilic balance of about 11 to about 16.
18. The kit of claim 13 , further comprising polyvinylpyrrolidone.
19. The kit of claim 13 , further comprising a component selected from the group consisting of a fluorosurfactant, an indicator dye, a preservative, a buffering agent, an enhancer of a LAMP-BART reaction or a qPCR reaction, and a combination of any two or more of the foregoing components.
20. The kit of claim 13 , wherein any one or more of the zirconium particles, the ferric iron, the polyvinylpyrrolidone, the non-ionic surfactant, the fluorosurfactant, the indicator dye, the preservative, the buffering agent, or the enhancer is disposed in an aqueous solution, wherein the aqueous solution has a pH of about 8.45 to 8.85.