Process for cell lysis and nucleic acid amplification
The present disclosure provides a nucleic acid amplification method. The method comprises forming an enrichment culture by contacting a sample with a nutrient medium having a formulation that does not include a phosphate buffer component; holding the enrichment culture for a period of time at a temperature that facilitates growth of a target microorganism; after holding the enrichment culture, forming an aqueous composition by mixing a first volume of the enrichment culture with a second volume of a lysis buffer; contacting the aqueous composition with an effective amount of a water-insoluble material that sequesters a substance that interferes with a polymerase-mediated nucleic acid amplification reaction; subjecting the aqueous composition to a thermal lysis process; and, after subjecting the aqueous composition to the thermal lysis process, subjecting a portion of the aqueous composition to a nucleic acid amplification process. A composition for a lysis buffer is also disclosed.
1. A nucleic acid amplification method, said method comprising:
forming an enrichment culture by contacting a sample with a nutrient medium having a formulation that does not include a phosphate buffer component, wherein the nutrient medium comprises a first buffer;
holding the enrichment culture for a period of time at a temperature that facilitates growth of a target microorganism;
after holding the enrichment culture, forming an aqueous composition by mixing a first volume of the enrichment culture with a second volume of a lysis buffer to form a third volume of the aqueous composition;
wherein mixing the first volume of the enrichment culture with the second volume of the lysis buffer comprises mixing an undiluted first volume of the enrichment culture with the second volume of the lysis buffer;
wherein the lysis buffer comprises a second buffer;
wherein the lysis buffer comprises an organic multivalent cation-chelating reagent, wherein the organic multivalent cation-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;
wherein the lysis buffer has a pH greater than 8.6 at 25° C.;
wherein the aqueous composition has a pH of about 8.45 to 8.85 at 25° C.;
wherein a combined concentration of the first buffer and the second buffer in the aqueous composition is at least about 15 mM;
wherein the ratio of the first volume to the second volume is greater than or equal to 3:1 and the ratio of the second volume to the third volume is less than or equal to 1:4;
contacting the aqueous composition with an effective amount of a water-insoluble material that sequesters a substance that interferes with a polymerase-mediated nucleic acid amplification reaction;
subjecting the aqueous composition to a thermal lysis process; and
after subjecting the aqueous composition to the thermal lysis process, subjecting a portion of the aqueous composition to an isothermal nucleic acid amplification process.
2. The method of claim 1 , wherein the lysis buffer further comprises ferric iron.
3. The method of claim 1 , wherein the lysis buffer further comprises a reagent selected from the group consisting of a nanoparticle dispersion stabilizer, a non-ionic surfactant having a hydrophilic-lipophilic balance of about 11 to about 16, polyvinylpyrrolidone, magnesium sulfate heptahydrate, a fluorosurfactant, an indicator dye, and a combination of any two or more of the foregoing reagents.
4. The method of claim 1 , wherein holding the enrichment culture for a period of time comprises holding the enrichment culture for a period of about 4 hours to about 24 hours.
5. The method of claim 1 , wherein the ratio of the first volume to the second volume is greater than or equal to 5:1 and the ratio of the second volume to the third volume is less than or equal to 1:6.
6. The method of claim 1 , wherein forming the aqueous composition by mixing the first volume of the enrichment culture with the second volume of the lysis buffer comprises forming the aqueous composition with the water-insoluble material that sequesters out of the aqueous composition a substance that interferes with a polymerase-mediated nucleic acid amplification reaction disposed therein.
7. The method of claim 1 , wherein the water-insoluble material comprises a plurality of zirconium oxide particles.
8. The method of claim 7 , wherein the plurality of zirconium oxide particles has a surface area of about 10 m 2 /L to about 600 m 2 /L in the third volume.
9. The method of claim 1 , wherein subjecting a portion of the aqueous composition to a nucleic acid amplification process comprises subjecting the portion of the aqueous composition to a loop-mediated isothermal amplification process.
10. The method of claim 1 , wherein forming the aqueous composition consists essentially of mixing the first volume of the enrichment culture with the second volume of the lysis buffer.
11. The method of claim 1 further comprising:
after subjecting the aqueous composition to the thermal lysis process and before subjecting the portion of the aqueous composition to the nucleic acid amplification process, rehydrating a dehydrated reagent for nucleic acid amplification using the portion of the aqueous composition.
12. The method of claim 1 , wherein the enrichment culture comprises peptone.