US-12618100-B2 - Rapid cellular lysis by reduction/oxidation reaction

US12618100B2US 12618100 B2US12618100 B2US 12618100B2US-12618100-B2

Abstract

Provided herein are methods for the rapid preparation of amplifiable nucleic acids from biological samples, which can be applied to various applications, such as, for example, point-of-care diagnostics, service laboratory diagnostics, and molecular biology applications. These methods can be performed in 15 minutes or less, and preferably in 5 minutes or less. For most applications, no further purification of nucleic acids is needed.

Inventors

Edward A. SEKINGER
Megan MARTINEZ
Mahima PANCHOLI
Kurt HOFFACKER

Assignees

LUMINEX CORPORATION

Dates

Publication Date: 20260505
Application Date: 20200817

Claims (15)

1 . A method for preparing amplifiable nucleic acids from a biological sample, the method comprising: (a) contacting the biological sample with a percarbonate salt, a nuclease suppressor, and a chelator to form a redox reaction composition; (b) incubating the redox reaction composition at a first temperature, wherein the first temperature is from 20° C. to 65° C., wherein the redox composition is incubated at the first temperature for 1 to 3 minutes; and (c) incubating the redox reaction composition at a second temperature, wherein the second temperature is from 60° C. to 100° C., wherein the redox reaction composition is incubated at the second temperature for 30 to 90 seconds, thereby obtaining amplifiable nucleic acids from the biological sample without purification of the nucleic acids following the redox reaction.
2 . The method of claim 1 , wherein the first temperature is from 35° C. to 60° C.
3 . The method of claim 1 , wherein the second temperature is from 70° C. to 95° C.
4 . The method of claim 1 , wherein the method further comprises agitating the redox reaction mixture by mechanical agitating or sonicating.
5 . The method of claim 1 , further comprising contacting the biological sample with beads.
6 . The method of claim 5 , wherein the beads are silica beads or glass beads.
7 . The method of claim 1 , wherein the percarbonate salt comprises sodium percarbonate.
8 . The method of claim 1 , wherein the nuclease suppressor comprises Proteinase K.
9 . The method of claim 1 , wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).
10 . The method of claim 1 , wherein the method further comprises amplifying at least a portion of the amplifiable nucleic acids.
11 . The method of claim 10 , wherein neither sodium bicarbonate nor sodium thiosulfate are added to the redox reaction composition prior to amplifying at least a portion of the amplifiable nucleic acids.
12 . The method of any of claim 10 , wherein no wash step is performed on the redox reaction composition prior to amplifying at least a portion of the amplifiable nucleic acids.
13 . The method of claim 1 , wherein the redox reaction composition does not contain a detergent.
14 . The method of claim 1 , wherein the method is performed in 15 minutes or less.
15 . The method of claim 1 , wherein the method is performed in 5 minutes or less.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Ser. No. 62/889,160, filed Aug. 20, 2019, the entire contents of which are incorporated herein by reference. BACKGROUND 1. Field The present invention relates generally to the field of molecular biology. More particularly, it concerns methods for rapid cellular lysis using a reduction-oxidation reaction in order to obtain amplifiable nucleic acids. 2. Description of Related Art PCR and RT-PCR reactions require an input of nucleic acids that are often obtained by purifying DNA/RNA from a solid phase surface. These methods can be time consuming due to the requirements of nucleic acid binding, washing, and eluting from the surface. Methods are needed that provide amplifiable nucleic acids on a shortened time scale. SUMMARY As such, provided herein are methods for fast cell lysis and nucleic acid preparation. The methods may be automated or performed manually. In some aspects, these methods do not use inhibitory reagents (e.g., certain denaturants, chaotropic agents, organic solvents) that would need to be washed away before performing DNA or RNA amplification. However, a certain amount of inhibitory reagents are tolerated in the amplification reaction. In one embodiment, provided are methods of obtaining amplifiable nucleic acids from a biological sample, the methods comprising: (a) forming a redox reaction composition by contacting the biological sample with a percarbonate salt, a nuclease suppressor, and a chelator; (b) incubating the redox reaction composition at a first temperature that is between 20° C. and 65° C.; and (c) incubating the redox reaction composition at a second temperature that is between 60° C. and 100° C. In some aspects, the first temperature is between about 25° C. and 65° C., 35° C. and 65° C., 45° C. and 65° C., 50° C. and 65° C., 20° C. and 60° C., 25° C. and 60° C., 30° C. and 60° C., 35° C. and 60° C., 45° C. and 60° C., 20° C. and 55° C., 25° C. and 55° C., 30° C. and 55° C., 35° C. and 55° C., 40° C. and 55° C., 20° C. and 50° C., 25° C. and 50° C., 30° C. and 50° C., 35° C. and 50° C., 20° C. and 45° C., 25° C. and 45° C., 30° C. and 45° C., 20° C. and 40° C., 25° C. and 45° C., 20° C. and 35° C., or any range derivable therein. In some aspects, the first temperature is about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., or 65° C. In some aspects, step (b) comprises incubating the redox reaction composition at the first temperature for between about 1 minute and about 3 minutes, for between about 1 minute and about 2 minutes, for between about 2 minutes and about 3 minutes, for between about 90 seconds and about 3 minutes, or any range derivable therein. In some aspects, step (b) comprises incubating the redox reaction composition at the first temperature for about 60 seconds, about 75 seconds, about 90 seconds, about 105 seconds, about 120 seconds, about 135 seconds, about 150 seconds, about 165 seconds, or about 180 seconds. In some aspects, the second temperature is between about 60° C. and 75° C., 60° C. and 80° C., 60° C. and 85° C., 60° C. and 90° C., 60° C. and 95° C., 65° C. and 80° C., 65° C. and 85° C., 65° C. and 90° C., 65° C. and 95° C., 65° C. and 100° C., 70° C. and 85° C., 70° C. and 90° C., 70° C. and 95° C., 70° C. and 100° C., 75° C. and 90° C., 75° C. and 95° C., 75° C. and 100° C., 80° C. and 95° C., 80° C. and 100° C., 85° C. and 100° C., or any range derivable therein. In some aspects, the second temperature is about 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., or 100° C. In some aspects, step (c) comprises incubating the redox reaction composition at the second temperature for between about 30 seconds and about 90 seconds, for between about 30 and 60 seconds, for between about 60 and 90 second, or any range derivable therein. In some aspects, step (c) comprises incubating the redox reaction composition at the second temperature for about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, or about 90 seconds. In some aspects, the methods further comprise agitating the redox reaction composition. In some aspects, agitating comprises mechanical agitating (e.g., vortexing) or sonication. In some aspects, the redox r