CLAIM OF PRIORITY

[0001] This application claims priority to U.S. Provisional Application No. 63/011,187 filed on April 16, 2020, and U.S. Provisional Application No. 63/062,486 filed on August 7, 2020, and each of which are incorporated herein by reference.

BACKGROUND

[0002] The extensive research and development taking place in the rapidly growing fields of molecular biology and biotechnology has increased the demand for detecting and isolating ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). For example, RNA and DNA isolation is necessary for molecular cloning of genes which are important in the medical and agricultural fields (e.g., insulin, growth hormone genes, and genes responsible for increased plant productivity). In addition, isolation and characterization of RNA and DNA are necessary for the early detection of virus and other pathogens. In many contexts it is necessary or desirable to isolate the RNA and DNA from the same biological sample. This is extremely difficult, if not impossible, when the tissue sample size is very small, for example in biopsies or in cheek swabs, and cannot be divided and used in separate isolation techniques for each component.

[0003] Nucleic acid extraction protocols can be broadly classified into silica based and non-silica based protocols. The existing silica and non-silica protocols cannot tolerate a water-wash to remove non-nucleic acid components and require an aqueous wash with some percentage of alcohol in it. Presence of alcohol in eluted nucleic acid solution inhibits polymerase chain reaction (PCR) and hence, typically both the protocols require a high speed spinning or other methods to remove residual alcohol and elution of nucleic acids In some cases, both the protocols require a high salt concentration with polyethylene glycol or an aqueous alcohol wash. The use of silica based matrix, high concentration of salts and aqueous alcohol, and repeated centrifugation puts a restriction on the elution of nucleic acids at hospitals and at point of care (POC) sites as these processes are cumbersome, time consuming, and expensive.

[0004] Therefore, there is a need to develop methods and compositions to isolate nucleic acids by a rapid, single-step process that is affordable and easy to use.

SUMMARY

[0005] Disclosed herein are methods and compositions that allow for rapid extraction and purification of RNAfrom the same biological sample. In an embodiment, the method comprising steps of:

(a) adding a first buffer to the sample containing RNA to obtain a mixture;

(b) adding a second buffer to the mixture; and

(c) centrifuging the mixture to obtain a supernatant containing the RNA.

[0006] In some embodiments, the first buffer comprises one or more anionic detergents, one or more ionic detergents, one or more chaotropic agents, and one or more chelating agents. Non-limiting examples of anionic detergents include alkyl sulfates, alkyl aryl sulfates, alkyl aryl sulfonates and alkyl sarcosinates. In some embodiments, preferred anionic detergents include sodium dedocyl sulphate (SDS), cholic acid, taurocholic acid, and cetyl ammonium sulfate, and combinations thereof. In some embodiments, the first buffer comprises one or more chaotropic agents selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. In some embodiments, the first buffer comprises one or more non-ionic detergents selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monooleate (Tween-80), Triton X-100, ethoxylated alkyl phenol (NP-40), Polyoxyethylene (20) cetyl ether (Brij 58), N,N-Bis[3-(D-gluconamido)propyl]-cholamide (bigCHAP) and combinations thereof. In some embodiments, the first buffer comprises chelating agents, such as EDTA and EGTA.

[0007] In an additional embodiment, the second buffer comprises one or more salts, one or more non-ionic detergents, one or more pH adjusting agents, hydroxide base, one or more ionic detergents, and one or more chaotropic agents. Non-limiting examples of salts include comprises potassium acetate, sodium chloride, lithium acetate, magnesium chloride, ammonium acetate, and combinations thereof. In some embodiments, the second buffer comprises non-ionic detergents selected from NP40, Tween 20, Triton X-100, and combinations thereof. In some embodiments, the second buffer comprises one or more chaotropic agents selected from guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, and combinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0008] FIGURE 1 depicts electrophoresis of DNAfrom an avirulent mycobacterium species. The DNA was extracted by the single-tube method disclosed herein and amplified with two different primer sets as indicated, and subjected to electrophoresis.

[00091 FIGURE 2 depicts amplification of mycobacterium DNA that was extracted by the single-tube method disclosed herein and compared with the DNA extracted with a competitor kit. The amplification was measured by SYBR Green assay. The single-tube method showed early amplification showing high efficiency of extraction and purity of DNA. G00101 FIGURE 3 depicts CT values of DNA samples extracted by single-tube extraction method and by a competitor kit. DNA amplification was detected with both undiluted and diluted DNA samples that were extracted by the single-tube extraction method. However, no amplification was detected with undiluted DNA samples that were extracted from the competitor’s kit suggesting the presence of inhibitors in the sample.

[0011] FIGURE 4 depicts the sensitivity of the single-tube extraction method in detecting the DNA of M. Tuberculosis. The table shows that even 3 copies of bacterial cells could be detected around 30 cycles, thus showing high efficiency of the method.

[0012] FIGURE 5 depicts the extraction efficiency of the DNA extracted from saliva using the single-tube extraction method and competitor kits.

[0013] FIGURE 6 depicts the extraction efficiency of the DNA extracted from buccal swab using the single-tube extraction method and competitor kits.

[0014] FIGURE 7 shows RNA extracted from human plasma that was spiked with known amount of fibroblast cells. The results show that the quality of RNA extracted by the single-tube extraction method was equivalent to that of the RNA extracted by Trizol.

[0015] FIGURE 8 shows that RNA extracted with the single tube extraction method showed better PCR efficiency when compared to RNA extracted with competitor’s kits or using Trizol.

DETAILED DESCRIPTION

[0016] As used herein, the term “about” when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. [0017] The transitional term “comprising,” which is synonymous with “including,”

“containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. In embodiments or claims where the term comprising is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.”

[0018] The “weight percent” disclosed herein may be weight-to-weight percent or weight-to-volume percent, depending upon the composition.

[0019] The isolation of DNA or RNA is an important step in many biochemical and diagnostic procedures. For example, the separation of nucleic acids from the complex mixtures in which they are often found is frequently necessary before other studies and procedures, such as detection, cloning, sequencing, amplification, hybridization, cDNA synthesis etc. can be undertaken. The presence of large amounts of cellular or other contaminating material, such as proteins or carbohydrates, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. In addition, DNA may contaminate RNA preparations and vice versa. Thus, methods for the isolation of nucleic acids from complex mixtures such as cells, tissues etc. are demanded, not only from the preparative point of view, but also in the many methods in use today which rely on the identification of DNA or RNA. such as diagnosis of microbial infections, forensic science, tissue and blood typing, detection of genetic variations etc. [0020] Additionally, a number of viruses with a significant impact on human health, including HIV, HCV, West Nile Virus, Equine Encephalitis Virus, coronavirus, and Ebola Virus have RNA genomes. The ability to rapidly and cleanly extract viral RNA from bodily fluids or tissues is important in virology research and infectious disease diagnostics and treatment. To obtain satisfactory results in these analysis methods, the use of RNA with high purity is required. Particularly in the RT-PCR, RNA analysis becomes difficult when DNA is present with RNA. Accordingly, it is desired that RNA is isolated is substantially pure that is not contaminated with DNA, proteins, lipids, carbohydrates, and the like that are present in cells.

[0021] Compositions and methods disclosed herein allow for the extraction and purification of RNA from the same sample, thus providing an advantage to users. The method of this invention is rapid, typically requiring only a few minutes to complete. Significantly, the RNA obtained by the method are of an adequate purity such that it is useful for clinical or other downstream uses, such as the use of reverse transcriptase, by itself or followed by the polymerase chain reaction amplification (RT-PCR), RNA blot analysis and in vitro translation. Advantageously, it is not necessary to process the sample extensively prior to use of this method and only simple equipment is required for performance of the method. No preliminary lysis or ethanol precipitation step is necessary before processing samples in accordance with the method of the invention.

[0022] Disclosed herein are buffer compositions to isolate RNA from a sample. In some embodiments, the first buffer (Buffer A) comprises one or more anionic detergents, one or more ionic detergents, one or more chaotropic agents, and one or more chelating agents. Non-limiting examples of anionic detergents include alkyl sulfates, alkyl aryl sulfates, alkyl aryl sulfonates and alkyl sarcosinates. In some embodiments, preferred anionic detergents include sodium dedocyl sulphate (SDS), cholic acid, taurocholic acid, and cetyl ammonium sulfate, and combinations thereof. The anionic detergents may be present in the first buffer at a concentration from about 1 mM to about 500 mM, about 1 mM to about 400 mM, about 1 mM to about 300 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, or about 1 mM to about 50 mM. Specific examples include about 1 mM, about 10 mM, about 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM and ranges between any of these values.

[0023] In some embodiments, the first buffer comprises one or more non-ionic detergents selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monooleate (Tween-80), Triton X-100, ethoxylated alkyl phenol (NP-40), Polyoxyethylene (20) cetyl ether (Brij 58), N,N-Bis[3-(D-gluconamido)propyl]-cholamide (bigCHAP) and combinations thereof. In some embodiments, the first buffer comprises one or more non-ionic detergents at a concentration from about 0.01 wt% to about 4 wt%, about 0.01 wt% to about 3 wt%, about 0.01 wt% to about 2.5 wt%, about 0.01 wt% to about 2 wt%, about 0.01 wt% to about 1.5 wt%, or about 0.01 wt% to about 1 wt% of the total weight of the buffer.

[0024] In some embodiments, the first buffer comprises one or more chaotropic agents selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. In some embodiments, the first buffer comprises one or more chaotropic agents at a concentration from about 1 mM to about 1000 mM, about 1 mM to about 700 mM, about 1 mM to about 500 mM, about 1 mM to about 400 mM, about 1 mM to about 300 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, or about 1 mM to about 20 mM. Specific examples include about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, about 250 mM, about 300 mM, about 1000 mM, and ranges between any of these values.

[0025] In some embodiments, the first buffer comprises chelating agents, such as EDTA and EGTA in concentration ranges from about 1 mM to about 100 mM, about 1 mM to about 70 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, or about 1 mM to about 20 mM.

[0026] In some embodiments, the first buffer optionally comprises a nucleic acid molecule that can act as a carrier RNA/DNA, such as poly-A RNA, salmon sperm DNA, or any other carrier RNA used in the art. The amount of nucleic acid that can be present in the first buffer may be from about 1 microgram/mL to about 1000 micrograms/mL, about 1 microgram/mL to about 500 micrograms/mL, about 1 microgram/mL to about 200 micrograms/mL, about 1 microgram/mL to about 100 micrograms/mL, about 1 microgram/mL to about 50 micrograms/mL.

[0027] In some embodiments, the first buffer optionally comprises a serine protease, such as proteinase K in the range from about 0.5 millirams/mL to about 20 milligrams/mL, about 0.5 milligrams/mL to about 10 milligrams/mL, or about 0.5 milligrams/mL to about 5 milligrams/mL.

[0028] In some embodiments, the first buffer optionally comprises an endonuclease, such as DNase I in a range from about 2 units/mL to about 50 units/mL, about 2 units/mL to about 40 units/mL, about 2 units/mL to about 30 units/mL, about 2 units/mL to about 20 units/mL, about 2 units/mL to about 10 units/mL, or about 2units/mL to about 5 units/mL. [0029] In some embodiments, the first buffer optionally comprises a ribonuclease inhibitor, such as RNase I in a range from about 1000 units/mL to about 20,000 units/mL, about 1000 units/mL to about 10,000 units/mL, about 1000 units/mL to about 5000 units/mL, or about 1000 units/mL to about 2000 units/mL.

[0030] In some embodiments, the first buffer consists of an anionic detergent, a chaotropic agent a carrier DNA, a serine protease, an endonuclease, and a ribonuclease inhibitor. In some embodiments, the anionic detergent is selected from sodium dedocyl sulphate (SDS), cholic acid, taurocholic acid, and cetyl ammonium sulfate, and combinations thereof. In some embodiments, the chaotropic agent is selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof.

[0031] In some embodiments, the first buffer consists of non-ionic detergent, chaotropic agent a carrier DNA, a serine protease, an endonuclease, and a ribonuclease inhibitor. In some embodiments, non-ionic detergents are selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monooleate (Tween-80), Triton X-100, ethoxylated alkyl phenol (NP-40), Polyoxyethylene (20) cetyl ether (Brij 58),

N,N-Bis[3-(D-gluconamido)propyl]-cholamide (bigCHAP) and combinations thereof. In some embodiments, the chaotropic agent is selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. [0032] In some embodiments, the first buffer consists of a chaotropic agent, a chelating agent a carrier DNA, a serine protease, an endonuclease, and a ribonuclease inhibitor. In some embodiments, the chaotropic agent is selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. In some embodiments, the chelating agent is selected from EDTA and EGTA.

[0033] In some embodiments, the first buffer consists of a non-ionic detergent, an anionic detergent, a chelating agent, a carrier DNA, a serine protease, an endonuclease, and a ribonuclease inhibitor. In some embodiments, non-ionic detergents are selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monooleate (Tween-80), Triton X-100, ethoxylated alkyl phenol (NP-40), Polyoxyethylene (20) cetyl ether (Brij 58), N,N-Bis[3-(D-gluconamido)propyl]-cholamide (bigCHAP) and combinations thereof. In some embodiments, the anionic detergent is selected from sodium dedocyl sulphate (SDS), cholic acid, taurocholic acid, and cetyl ammonium sulfate, and combinations thereof. In some embodiments, the chelating agent is selected from EDTA and EGTA.

[0034] In some embodiments, the first buffer (Buffer A) has the following composition:

[0035] In other embodiments, the first buffer (Buffer A) has a composition as follows: [0036] In some embodiments, the second buffer (Buffer B) comprises one or more salts, one or more non-ionic detergents, one or more pH adjusting agents, hydroxide base, one or more ionic detergents, one or more chelating agents, and one or more chaotropic agents. Non-limiting examples of salts include ammonium acetate, ammonium phosphate, ammonium sulfate, diammonium hydrogen phosphate, lithium chloride, lithium sulfate, lithium acetate, magnesium sulfate, magnesium chloride, potassium chloride, potassium citrate, potassium phosphate, potassium acetate, sodium acetate, sodium chloride, sodium citrate, sodium phosphate, and sodium sulfate, and combinations thereof. In some embodiments, the second buffer comprises one or more salts at a concentration from about 1 mM to about 4000 mM, about 1 mM to about 3500 mM, about 1 mM to about 3000 mM, about 1 mM to about 2500 mM, about 1 mM to about 2000 mM, or about 1 mM to about 1000 mM. Specific examples include about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 50 mM, about 100 mM, about 300 mM, about 1000 mM, and ranges between any of these values. In some embodiments, the second buffer comprises potassium acetate, potassium chloride, sodium chloride, lithium acetate, magnesium chloride, magnesium sulphate, and combinations thereof.

[0037] In some embodiments, the second buffer comprises one or more non-ionic detergents selected from polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monooleate (Tween-80), Triton X-100, ethoxylated alkyl phenol (NEMO), Polyoxyethylene (20) cetyl ether (Brij 58),

N,N-Bis[3-(D-gluconamido)propyl]-cholamide (bigCHAP) and combinations thereof. In some embodiments, the second buffer comprises one or more non-ionic detergents at a concentration from about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2.5 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 1.5 wt%, or about 0.1 wt% to about 1 wt% of the total weight of the buffer. In some embodiments, the second buffer comprises non-ionic detergents selected from NP40, Tween 20, Triton X-100, and combinations thereof.

[0038] In some embodiments, the second buffer comprises one or more one or more chaotropic agents selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. In some embodiments, the second buffer comprises one or more chaotropic agents at a concentration from about 10 mM to about 2000 mM, about 10 mM to about 1500 mM, about 10 mM to about 1200 mM, about 10 mM to about 1000 mM, about 10 mM to about 700 mM, or about 10 mM to about 500 mM. Specific examples include about 10 mM, about 50 mM, about 75 mM, about 100 mM, about 120 mM, about 150 mM, about 500 mM, about 100 mM, about 2000 mM and ranges between any of these values.

[0039] In some embodiments, the second buffer comprises one or more pH adjusting agents, such as Tris base, a phosphate, a citrate, an acetate, and the like. The pH adjusting agents may be present in the concentration from about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, or about 1 mM to about 10 mM.

[0040] In some embodiments, the second buffer comprises hydroxide base, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, magnesium hydroxide, and the like. The hydroxide bases may be present in the concentration from about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, or about 1 mM to about 10 mM. [0041] In some embodiments, the second buffer comprises one or more anionic detergents, such as sodium dedocyl sulphate (SDS), cholic acid, taurocholic acid, and cetyl ammonium sulfate, and combinations thereof. The anionic detergents may be present in the second buffer at a concentration from about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, or about 100 mM to about 150 mM.

[0042] In some embodiments, the second buffer comprises one or more chelating agents, such as EDTA and EGTA in concentration ranges from about 1 mM to about 100 mM, about 1 mM to about 70 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, or about 1 mM to about 20 mM.

[0043] In some embodiments, the second buffer consists of a pH adjusting agent, a chelating agent, one or more salts, and a chaotropic agent. In some embodiments, pH adjusting agent is selected from Tris base, a phosphate, a citrate, an acetate, and combinations thereof. In some embodiments, the chelating agent is selected from EDTA and EGTA. In some embodiments, the chaotropic agent is selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, aminoguanidine HC1, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, urea, thiourea, and combinations thereof. In some embodiments, the one or more salts are selected from potassium acetate, potassium chloride, sodium chloride, lithium acetate, magnesium chloride, magnesium sulphate, and combinations thereof.

[0044] In some embodiments, the second buffer consists of a pH adjusting agent and a salt. In some embodiments, pH adjusting agent is selected from Tris base, a phosphate, a citrate, an acetate, and combinations thereof. In some embodiments, the salt is selected from potassium acetate, potassium chloride, sodium chloride, lithium acetate, magnesium chloride, magnesium sulphate, and combinations thereof.

[0045] In some embodiments, the second buffer (Buffer B) has the following composition:

[0046] In some embodiments, the second buffer (Buffer B) has the following composition: [0047] The present disclosure relates to a method for isolation of RNA from a sample, the method comprising steps of:

(a) adding a first buffer (Buffer A) disclosed herein to the sample containing RNA to obtain a mixture;

(b) adding a second buffer (Buffer B) disclosed herein to the mixture; and

(c) centrifuging the mixture to obtain a supernatant containing the RNA.

[0048] In some embodiments, the steps recited above can be interchanged. For example, the method comprises:

(a) adding the Buffer B disclosed herein to the sample containing RNA to obtain a mixture;

(b) adding the Buffer A disclosed herein to the mixture; and

(c) centrifuging the mixture to obtain a supernatant containing the RNA.

[0049] In some embodiments, the sample from which the RNA is to be isolated is a biological or non-biological sample. In some embodiments, the biological sample is selected from a group comprising blood, sputum, serum, saliva, urine, semen, cell cultures, nasal swabs, cheek swabs, or tissue extracts. Biological samples also include plant materials, eukaryotes, bacteria, viruses, fungi, and prokaryotes. In some embodiments, non-biological samples include solvents, seawater, industrial water samples, food samples, and environmental samples such as soil or water.

[0050] In some embodiments, solubilization of the sample is carried out by a physical method that uses a mortar, ultrasound, microwave, homogenizer, or the like, a chemical method that uses a surface active agent, protein denaturant, or the like, or a biochemical method utilizing a proteinase, and by a method in combination of these methods. [0051] In some embodiments, the method comprises adding the first buffer to the sample containing RNA. Adding the first buffer to the sample containing RNA comprises mixing the first buffer disclosed herein and the sample by any method known in the art, such as shaking, vortexing, pipetting up and down, rocking, and the like. Typically, it is desirable to incubate the sample with the first buffer described herein for a period of time at a desired temperature. Typically, a suitable incubation time is a period of or greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 minutes. In some embodiments, a suitable incubation time is a period of or less than about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 minutes. In some embodiments, the mixture of the sample and the first buffer is incubated for about 5 minutes at room temperature. Typically, “room temperature” or “ambient temperature” refers to a temperature with the range of about 20-25° C., for example, about 20° C., 21° C., 22° C., 23° C., 24° C., or 25° C. In some embodiments, the mixture may also be incubated above room temperature, such as about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C. In some embodiments, the mixture may be incubated at two different temperatures, such as 50-65 °C for 5 minutes followed by 80-95 °C for 5 minutes. In some embodiments, the mixture of sample and the first buffer may undergo mixing or shaking during the entire incubation period. In some embodiments, the mixing and incubation process disclosed above can also be applied when the method steps are interchanged, i.e., when the sample and the second buffer are mixed.

[0052] In some embodiments, the method comprises adding the second buffer to the mixture comprising the first buffer and the sample. In some embodiments, adding the second buffer to the mixture comprises adding the second buffer described herein to the mixture comprising the sample and first buffer. The second buffer may be added to the mixture and mixed by any method known in the art, such as shaking, vortexing, pipetting up and down, rocking, and the like.

[0053] In some embodiments, the method further comprises incubating the mixture on ice or at 4° C for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 minutes. This step would result in cloudy appearance of the mixture.

[0054] In some embodiments, the method comprises an optional step of centrifuging the mixture to obtain a supernatant containing the RNA. Centrifugation may be performed by using any centrifuge or spinning equipment known in the art, at about 6000 g to about 20,000 g for about 2 minutes to about 20 minutes, at 4° C. The centrifugation step results in formation of the precipitate and the supernatant, and the supernatant comprises the RNA which can be used for downstream processing.

[0055] The extracted RNA using the methods described herein can be ready to be used in a PCR or RT-PCR. Other applications include recovery of RNA from a clinical sample for archiving, storage, further biochemical and molecular biology use etc. The extracted RNA can be used for any biochemical or molecular biological application, which a person in the state of the art will discover from time to time. In some embodiments, the extracted RNA may be used to detect infection of a virus including but not limited to HIV, HCV, influenza virus, West Nile Virus, Equine Encephalitis Virus, coronavirus, SARS, MERS, SARS-CoV-2, and Ebola Virus. In some embodiments, the extracted DNA may be used to detect pathogens, such as E. coli, Salmonella, Pseudomonas spp., Staphylococcus aureus, and M. Tuberculosis.

[0056] All of the steps disclosed herein can be performed rapidly, in succession, and in a single container without the need for specialized equipment such as homogenizers, sonicators, or without use of solvents such as ethanol precipitation and phenol extraction or use of complex processes, such as magnetic beads and silica-based extraction columns. The method is adaptable to automated platforms for processing large numbers of samples in serial or parallel fashion. The methods disclosed herein can be performed in a single sample tube, such as microcentrifuge tube, a spin tube, or in multi -well plates such as a 96-well plate or a 384-well plate, and avoid the need to transfer any of the contents to multiple tubes during the processes and reduces errors and contamination. Further, the method can be carried out on ice or on benchtop at room temperature or at 65 degrees centigrade to remove inhibitors. In addition, the centrifugation step is optional to remove debris that may interfere with downstream application.

EXAMPLE 1: Single-tube extraction of RNA from plasma

[0057] To 100 ul of plasma, lOOul of first buffer was added and mixed by pipetting up and down 5 times. Immediately, about 50 ul of second buffer was added to the mixture and mixed by pipetting up and down. The mixture was spun briefly at 12000 g for 5 minutes. The supernatant was collected and about 4 ul was used for RNA applications.

[0058] First Buffer composition - SDS 200 mM and Urea 40 mM [0059] Second buffer composition - Potassium Acetate 75 mM, NaCl 1500 mM, Lithium Acetate 85 mM, NP40 1%, KC1 210 mM, MgCl ₂ 70 mM, Tween 0.2%, Triton 0.4%, Ammonium Acetate 150 mM, and Guanidine HCL 1200 mM.

EXAMPLE 2: Single-tube extraction of RNA from cell pellet

[0060] About 100 pL of cell pellet in a clean centrifuge tube was mixed with 100 pL of first buffer of Example 1 and mixed well. The mixture was incubated for 15 minutes at 65 degrees centigrade with shaking around 900 rpm /min. About 100 pL of second buffer of Example 1 was added and immediately pipette it up and down few times to ensure that the solution was mixed well. The mixture was kept on ice for 2 min until the cloudiness appeared. The mixture was centrifuged at 4°C at 12000g for 20 min. The supernatant was collected and was ready to use for RT-PCR.

EXAMPLE 3: Single-tube extraction of RNAfrom swab sample

[0061] About 100 L of first buffer (250 mM Guanidine hydrochloride + 25 mM EGTA

+ 50 ng of ribonucleic acid +5 ng of carrier RNA) was mixed with 100 L of swab sample. The mixture was heated between 55 C-65 C for 5-10 minutes. About 100 L of second buffer (8.5 mM NaOH + 5 mM Tris) was added to this mixture, mixed, and centrifuged at 14,000rpm for 5-10 minutes. The supernatant was collected and was ready to use for RT-PCR.