RAPID CELLULAR LYSIS BY REDUCTION/OXIDATION REACTION

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.

Claims

1-28. (canceled)

29. A method of obtaining 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.; and (c) incubating the redox reaction composition at a second temperature, wherein the second temperature is from 60 C. to 100 C.

30. The method of claim 29, wherein the redox composition is incubated at the first temperature for 1 to 3 minutes.

31. The method of claim 29, wherein the redox reaction composition is incubated at the second temperature for 30 to 90 seconds.

32. The method of claim 29, wherein the first temperature is from 35 C. to 60 C.

33. The method of claim 29, wherein the second temperature is from 70 C. to 95 C.

34. The method of claim 29, wherein the method further comprises agitating the redox reaction mixture by mechanical agitating or sonicating.

35. The method of claim 29, further comprising contacting the biological sample with beads.

36. The method of claim 35, wherein the beads are silica beads or glass beads.

37. The method of claim 29, wherein the percarbonate salt comprises sodium percarbonate.

38. The method of claim 29, wherein the nuclease suppressor comprises Proteinase K.

39. The method of claim 29, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).

40. The method of claim 29, wherein the method further comprises amplifying at least a portion of the amplifiable nucleic acids.

41. The method of claim 40, 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.

42. The method of any of claim 40, wherein no wash step is performed on the redox reaction composition prior to amplifying at least a portion of the amplifiable nucleic acids.

43. A composition comprising a percarbonate salt, a nuclease suppressor, and a chelator, wherein the composition is in a dried form.

44. The composition of claim 43, wherein the percarbonate salt comprises sodium percarbonate.

45. The composition of claim 43, wherein the nuclease suppressor comprises proteinase K.

46. The composition of any of claim 43, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA).

47. The composition of any of claim 43, wherein the composition further comprises beads.

48. The composition of any of claim 43, wherein the composition is essentially free of ascorbic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIG. 1Rapid Lysis of Samples in Various Media Using the Redox Protocol. In each pair of bars, the left bar represents the Redox Protocol and the right bar represents the ARIES benchtop.

DETAILED DESCRIPTION

[0030] Provided herein are methods for the rapid preparation of nucleic acids from biological samples, which can be applied to various point-of-care diagnostic 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. As such, the methods lack reagents that are inhibitory to PCR and RT-PCR, such as, for example, strong denaturants or chaotropic agents (e.g., guanidinium isothiocyanate (GITC)) and/or organic solvents (e.g., isopropyl alcohol (IPA)). Nevertheless, a certain amount of inhibitory reagents may be present. However, the concentration of any inhibitory reagents will be too low to be significantly inhibitory to a PCR or RT-PCR reaction and thus are tolerated in the amplification reaction.

[0031] Provided here is a general description of a protocol according to one embodiment of the invention: Prior to the initial redox reaction, beads (e.g., silica low-binding beads) are added to the biological sample. In some aspects, 10-50 milligrams of silica low binding beads, such as 100 micron beads may be used. The volume of the biological sample can be varied at least depending on the amount of material available. The redox reaction comprises adding sodium percarbonate (e.g., at least about 35 mM final concentration), a chelator (e.g., at least about 0.1 mM final concentration of EDTA), and a nuclease suppressor (e.g., such as a Proteinase) to the biological sample. The sodium percarbonate generates H.sub.2O.sub.2, which damages cells, thereby making them more susceptible to lysis. The chelator (e.g., EDTA) is added to the sample to inhibit DNase. The nuclease suppressor (e.g., Proteinase K) is added to the sample to inhibit RNase. The redox reaction is incubated for about two minutes at about 60 C. The sample is then heated for about one minute at about 80 C., and then sonicated for about one minute. Finally, the lysate is diluted at least 1:4 in a pH buffered solution (e.g., a Tris buffered solution having a pH of about 6.5 to 7.5) and added directly to an amplification reaction, such as, for example, a RT-PCR reaction master mix.

[0032] By biological sample is meant a sample comprising any biological material which samples can be prepared for use in the method of this invention. This includes, but is not limited to, bacterial cultures, yeast cultures, cells infected with virus, isolated virus, tissue cultures, cell lines, foods contaminated with bacteria, blood, serum, patient samples, urine, and other body fluids.

[0033] By lysis of a cell is intended the disruption, rupture, poration, permeabilization, digestion, or break down of the cell membrane (and cell wall, where applicable) such that the nucleic acid components of the cell can be released into the external solution. According to the invention, the cell membrane need not be completely disrupted, ruptured, permeabilized or digested in order to effect the release of the nucleic acids.

[0034] By chelator is meant chemical compounds that react with metal ions to form a stable, water-soluble complex. The chelator is typically provided in a concentrated aqueous solution that is pH-adjusted with small amounts of concentrated acid or base, as appropriate, to achieve a pH in the physiological range. Alternatively, any of several well-known buffers can be used to adjust the pH. The chelator will have a pH of about pH 7.0 to about pH 8.0, preferably a pH of about 7.5+/0.1 pH units. The chelator may be ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether) tetraacetic acid (EGTA), or their salts; more preferably, the chelating agent is EDTA. The terms EDTA and EGTA will be used to refer both to the acid and the salt form, and either form may be used in the present invention, although the salt forms are preferred.

[0035] By nuclease suppressor is meant an agent that inhibits the function of any nucleases present in a biological sample. Such nuclease suppressors may function by degrading any nuclease enzymes present. For example, the nuclease suppressor may be a non-specific protease, such as, for example, Proteinase K. Incubating the biological sample with Proteinase K will digest any protein present in the sample, including nucleases. The protease can be added to the sample after the chelator or simultaneously with the chelator. In order to accomplish digestion of the protein present, the biological sample with the added protease will be incubated at a temperature (e.g., between about 50 C. and about 65 C., preferably about 60 C.) and for a time (e.g., about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes) sufficient to allow the protease to work. These conditions are well known and readily determined by one of ordinary skill in the art.

[0036] As used herein nucleic acid means either DNA or RNA, either single-stranded or double-stranded.

[0037] As used herein, amplification or amplifying refers to the in vitro production of additional copies of a target nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies known in the art. The term amplification reaction refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These may include enzymes (e.g., a thermostable polymerase), aqueous buffers, salts, amplification primers, target nucleic acid, nucleoside triphosphates, and optionally, at least one labeled probe and/or optionally, at least one agent for determining the melting temperature of an amplified target nucleic acid (e.g., a fluorescent intercalating agent that exhibits a change in fluorescence in the presence of double-stranded nucleic acid).

[0038] The term PCR encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 L. Reverse transcription PCR, or RT-PCR, means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., U.S. Pat. No. 5,168,038. Real-time PCR means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., U.S. Pat. No. 5,210,015 (Taqman); U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); U.S. Pat. No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). Nested PCR means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. Initial primers in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and secondary primers mean the one or more primers used to generate a second, or nested, amplicon. Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture. Usually, distinct sets of primers are employed for each sequence being amplified. Quantitative PCR means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen.

[0039] The amplification methods described herein may include real-time monitoring or continuous monitoring. These terms refer to monitoring multiple times during a cycle of PCR, preferably during temperature transitions, and more preferably obtaining at least one data point in each temperature transition. The term homogeneous detection assay is used to describe an assay that includes coupled amplification and detection, which may include real-time monitoring or continuous monitoring.

[0040] The RT-PCR-grade nucleic acids can be detected and/or analyzed by any conventional detection technique, including e.g., amplification techniques such as PCR, TMA, NASBA, RT-PCR, optionally followed by sequencing analysis, if it is desirable for determination of, e.g., the types, species, and strains of microorganism detected.

EXAMPLES

[0041] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1Rapid Lysis Using the <15 Minute Redox Protocol

[0042] Multiple DNA/RNA targets were assayed using a pathogen cocktail. The pathogen cocktail was comprised of RNA viruses (Flu A and Flu B), a gram-negative bacteria (Bordetella parapertussis), a gram-positive bacteria (S. aureus), and a yeast (C. albicans).

[0043] Prior to the initial redox reaction, about 30 milligrams of 100 micron silica low-binding beads were added to 100 L of the pathogen cocktail. The redox reaction comprised adding sodium percarbonate (35 mM final concentration), proteinase K (0.04 mg), and EDTA (0.1 mM final concentration) to the pathogen cocktail. The redox reaction was incubated for about two minutes at about 60 C. The sample was heated for about one minute at about 80 C., and then sonicated for about one minute. Finally, the pathogen cocktail was diluted 1:4 in 50 mM Tris (pH 7) and added directly to an RT-PCR reaction master mix.

[0044] As a process control, pathogen cocktail was subjected to identical conditions except that only water was added instead of the redox reaction components. As a positive control, the pathogen cocktail was extracted and purified using a Luminex ARIES Benchtop system. The results are provided in Table 1.

TABLE-US-00001 TABLE 1 Real-time RT-PCR results for the 15 Minute Redox Protocol Pathogen Method Avg Ct StdDev Ct Avg Tm Positivity B. ARIES 32.8 0.2 89.4 100% parapertussis Benchtop Redox 29.0 0.2 90.3 100% Reaction Process 32.8 0.5 90.5 100% Control C. albicans ARIES 35.4 0.8 80.9 100% Benchtop Redox 30.8 0.2 81.7 100% Reaction Process 37.8 1.8 82.1 100% Control S. aureus ARIES 34.7 0.3 78.3 100% Benchtop Redox 34.5 0.2 78.9 100% Reaction Process 34.4 0.9 78.7 100% Control Flu A ARIES 29.5 0.4 82.9 100% Benchtop Redox 29.4 0.3 83.6 100% Reaction Process 29.3 0.4 83.6 100% Control Flu B ARIES 26.2 0.8 79.0 100% Benchtop Redox 28.3 0.2 79.6 100% Reaction Process 32.0 0.5 79.5 100% Control

[0045] As illustrated in Table 1, the redox reaction resulted in lower target Cts, save Flu B, which is indicative of better performance than the other two methods tested, e.g., ARIES benchtop and process control. Additionally, the standard deviations were less than 1, which demonstrates good reproducibility with the redox reaction process. Avg Ct: average Ct from 6 data points (2 biological replicates and 3 technical replicates=6 Ct values); StdDev Ct: variability determined for the 6 data points; Avg Tm: average temperature required to melt the PCR product; Positivity: percentage of successful target detection from 6 amplification reactions, e.g., 6 out of 6=100%.

Example 2Rapid Lysis Using the 5 Minute Redox Protocol

[0046] Multiple DNA/RNA targets were assayed using a pathogen cocktail. The pathogen cocktail was comprised of an RNA virus (Flu A/B), a gram-negative bacteria (Bordetella parapertussis), a gram-positive bacteria (S. aureus), and a yeast (C. albicans).

[0047] Prior to the initial redox reaction, about 30 milligrams of 100 micron silica low-binding beads were added to 100 L of the pathogen cocktail. The redox reaction comprised adding sodium percarbonate (35 mM final concentration), proteinase K (0.04 mg), and EDTA (0.1 mM final concentration) to the pathogen cocktail. The redox reaction was incubated for about two minutes at about 60 C. After the redox reaction, the sample was heated for about minute at about 80 C., and then sonicated for about 60 seconds. Finally, the pathogen cocktail was diluted 1:4 in 50 mM Tris (pH 7) and added directly to an RT-PCR reaction master mix.

[0048] As a process control, pathogen cocktail was subjected to identical conditions except that only water was added instead of the redox reaction components. As a positive control, the pathogen cocktail was analyzed using a Luminex ARIES Benchtop system. The results are provided in Table 2.

TABLE-US-00002 TABLE 2 Real-time RT-PCR results for the 5 Minute Redox Protocol Pathogen Method Avg Ct StdDev Ct Avg Tm Positivity B. ARIES 31.6 0.1 90.0 100% parapertussis Benchtop Redox 28.9 0.3 90.0 100% Reaction Process 30.5 0.5 90.3 100% Control C. albicans ARIES 32.9 0.7 81.6 100% Benchtop Redox 29.9 0.4 81.7 100% Reaction Process 36.5 0.9 81.7 50% Control S. aureus ARIES 33.8 1.0 78.1 100% Benchtop Redox 32.3 0.5 78.5 100% Reaction Process 33.0 0.2 79.0 100% Control Flu A ARIES 30.3 0.5 82.9 100% Benchtop Redox 31.9 0.8 83.1 100% Reaction Process 31.6 0.3 83.6 100% Control Flu B ARIES 29.4 0.5 79.1 100% Benchtop Redox 29.6 0.7 79.3 100% Reaction Process 34.2 0.5 79.6 100% Control RSV ARIES 29.2 0.2 75.7 100% Benchtop Redox 31.2 0.6 75.8 100% Reaction Process 38.8 1.3 0% Control

[0049] As displayed in Table 2, the redox reaction resulted in lower target Cts for DNA targets, but RNA target Cts were slightly higher than ARIES benchtop. Avg Ct: average Ct from 9 data points (3 biological replicates and 3 technical replicates=9 Ct values); StdDev Ct: variability determined for the 9 data points; Avg Tm: average temperature required to melt the PCR product; Positivity: percentage of successful target detection from 9 amplification reactions, e.g., 9 out of 9=100%.

Example 3Rapid Lysis of Samples in Various Media Using the 5 Minute Redox Protocol

[0050] The same experiment as described in Example 1 was performed, except that the pathogen cocktail also contained media. Three different types of media were tested to determine if they inhibited the 5 minute redox lysis protocol. The media tested were eSwab (Copan), MicroTest M5 (Remel, Thermo Fisher), and UTM (Copan). As a positive control, the samples were also analyzed using a Luminex ARIES Benchtop system. The graph in FIG. 1 shows that none of the media inhibited the 15 minute redox lysis protocol, relative to the positive control.

Example 4Rapid Lysis of Clinical Samples Using the 5 Minute Redox Protocol

[0051] The same experiment as described in Example 2 was performed, except that the samples tested were clinical samples known to be positive for the pathogens indicated in Table 3. Four different pathogens were tested: Flu A, Flu B, RSV, and C. albicans. As a positive control, the samples were analyzed using a Luminex ARIES Benchtop system. The results are provided in Table 3.

TABLE-US-00003 TABLE 3 Real-time RT-PCR results for the 5 Minute Redox Protocol using Clinical Samples Pathogen Method Avg Ct StdDev Ct Avg Tm Positivity C. albicans ARIES 20.7 0.5 81.6 100% Benchtop Redox 20.6 0.2 82.0 100% Reaction Flu A - ARIES 34.7 n/a 82.6 100% Sample 1 Benchtop Redox 36.1 0.4 83.0 100% Reaction Flu A - ARIES 34.1 n/a 83.0 100% Sample 2 Benchtop Redox 34.2 0.1 83.5 100% Reaction Flu B - ARIES 30.1 n/a 80.4 100% Sample 1 Benchtop Redox 29.7 0.5 80.5 100% Reaction Flu B - ARIES 30.9 n/a 80.8 100% Sample 2 Benchtop Redox 31.5 0.3 81.0 100% Reaction RSV - ARIES 26.0 n/a 75.9 100% Sample 1 Benchtop Redox 27.5 0.5 76.3 100% Reaction RSV - ARIES 30.9 n/a 75.6 100% Sample 2 Benchtop Redox 33.9 0.5 76.4 100% Reaction

[0052] Table 3 highlights the positivity testing of clinical samples when using the redox reaction, relative to the reference method of ARIES benchtop. Avg Ct: average Ct from 3 data points (1 biological extraction and 3 technical replicates=3 Ct values); StdDev Ct: variability determined for the 3 data points; Avg Tm: average temperature required to melt the PCR product; Positivity: percentage of successful target detection from 3 amplification reactions, e.g., 3 out of 3=100%.

[0053] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.