Composition, method and kit for cell lysis and nucleic acid extraction and method for molecular diagnosis

Abstract

This disclosure relates to a composition, a method and kit for cell lysis and nucleic acid extraction, and a molecular diagnostic method: which can minimize the time required for molecular diagnostics by using a composition containing a ribonuclease inhibitor as a solution for nucleic acid extraction and performing a polymerase chain reaction after heating to a specific temperature without separate purification and elution processes, and can reduce the cost of molecular diagnostics by minimizing the use of dedicated equipment and consumables for extraction.

Description

[Technical Field]

[0001] This application claims the benefit of Korean Patent Application No. 10-2021-0101536, filed on August 2, 2021, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety.

[0002] This disclosure relates to a composition for cell lysis and nucleic acid extraction, a nucleic acid extraction method using said composition, and a molecular diagnostic method using said composition. More specifically, it relates to the following composition for cell lysis and nucleic acid extraction, a nucleic acid extraction method using said composition, and a molecular diagnostic method using said composition: it can minimize the time required for molecular diagnostics by using a composition containing a ribonuclease inhibitor as a solution for nucleic acid extraction and performing a polymerase chain reaction after heating to a specific temperature without separate purification and elution processes, and can reduce the cost of molecular diagnostics by minimizing the use of dedicated equipment and consumables for extraction. [Previous Technology]

[0003] In recent years, due to the analysis of the causes of diseases at the gene level based on the results of human genome research, the demand for manipulating and biochemically analyzing biological samples for the treatment or prevention of human diseases has been gradually increasing. Furthermore, in various fields including new drug development, pre-testing for viral or bacterial infections, and forensic medicine, in addition to diagnosing diseases, there is a need for techniques to extract and analyze nucleic acids from biological samples or cell-containing samples.

[0004] At the same time, molecular diagnosis is usually performed by extracting nucleic acids (which are deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) containing genetic information) from the saliva or blood of a person suspected of being infected with a virus or bacteria, and amplifying the extracted nucleic acids to check whether the person is infected with the disease.

[0005] Figure 1 is a flowchart illustrating a molecular diagnostic method performed using polymerase chain reaction (PCR) according to conventional techniques. Referring to Figure 1, a molecular diagnostic method according to conventional techniques typically includes: obtaining a sample (step S10); extracting nucleic acids containing genetic information from the sample (step S30); amplifying the extracted nucleic acids by polymerase chain reaction (steps S70 and S90); and analyzing the amplification results. To extract nucleic acids, a lysis process (step S31), an elution process (step S33), and a purification process (step S50) are required. However, the lysis and purification processes used for nucleic acid extraction require specialized extraction equipment and consumables (e.g., plastic tools, magnetic beads, or solutions).

[0006] When nucleic acids are extracted using the aforementioned traditional methods, high-purity nucleic acids can be obtained. However, the problem is that the extraction process takes a long time, indicating that traditional methods are not suitable for diagnosis or testing in emergency situations or emergency rooms. In addition, when traditional methods are applied to situations where national prevention systems are needed due to the rapid spread of viruses, the diagnostic cost becomes high because specialized equipment and consumables must be used continuously for extraction.

[0007] Therefore, in order to solve the above problems, it is urgent to develop a technology that can use specific components to directly carry out polymerase chain reaction after cell lysis without purification or elution processes. [Summary of the Invention]

[0008] One object of this disclosure is to provide a composition for cell lysis and nucleic acid extraction, and a molecular diagnostic method using said composition, by using a composition containing specific components in a process of lysing cells to extract nucleic acids from the cells and heating a mixture containing lysed cells, said method can omit separate processes of purifying and eluting the solution containing lysed cells, and can use the solution containing lysed cells to perform polymerase chain reaction.

[0009] However, the purpose of this disclosure is not limited to the above-mentioned purposes, and those skilled in the art will clearly understand from the following description other purposes not mentioned herein.

[0010] One embodiment of this disclosure provides a composition for cell lysis and nucleic acid extraction, the composition comprising: a ribonuclease inhibitor; and a buffer.

[0011] According to one embodiment of the present disclosure, the ribonuclease inhibitor may include an inhibitor of ribonuclease A.

[0012] According to one embodiment of the present disclosure, the ribonuclease inhibitor may be derived from a protein.

[0013] According to one embodiment of the present disclosure, the pH of the buffer may be 6.0 to 9.0.

[0014] According to one embodiment of the present disclosure, the buffer may contain any one selected from glycerol, hydroxyethyl piperazine ethane sulfonic acid (HEPES), dithiothreitol (DTT), potassium chloride, and combinations thereof.

[0015] Another embodiment of this disclosure provides a method for cell lysis and nucleic acid extraction, the method comprising: a step of preparing a mixture by adding a sample containing nucleic acid to a composition for cell lysis and nucleic acid extraction; a first heating step of maintaining the mixture at a temperature of 25°C to 45°C; and a second heating step of maintaining the heated mixture at a temperature of 75°C to below 100°C.

[0016] According to one embodiment of the present disclosure, the first heating step and the second heating step can each be performed for 1 minute to 30 minutes.

[0017] According to one embodiment of the present disclosure, based on a 30 μL volume of mixture, the concentration of ribonuclease inhibitor in the mixture can be from 7.5 units / reaction to 60.0 units / reaction.

[0018] Another embodiment of this disclosure provides a molecular diagnostic method, the method comprising the following steps: adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by the method for cell lysis and nucleic acid extraction; and amplifying the extracted nucleic acids by polymerase chain reaction.

[0019] According to one embodiment of the present disclosure, the composition for cell lysis and nucleic acid extraction enables the minimization of time required for nucleic acid amplification-based molecular diagnostic processes by omitting a separate process for purifying and eluting the solution containing cells, and using a solution containing lysed cells for polymerase chain reaction.

[0020] A method for cell lysis and nucleic acid extraction according to an embodiment of the present disclosure can improve the accuracy of molecular diagnostics by inactivating factors that inhibit the accuracy of polymer chain reactions through heating during the nucleic acid extraction process.

[0021] A molecular diagnostic method according to an embodiment of the present disclosure can reduce the cost of molecular diagnostics by minimizing the use of specialized equipment and consumables for extraction.

[0022] The effects disclosed herein are not limited to those described above, and those skilled in the art will clearly understand from this specification and the accompanying drawings the effects not mentioned herein.

Implementation Method

[0024] Throughout this specification, it should be understood that when any part is referred to as "including" any component, it does not exclude other components, but may further include other components, unless otherwise stated.

[0025] Throughout this specification, "A and / or B" means "A and B" or "A or B".

[0026] This disclosure will be explained in more detail below.

[0027] One embodiment of this disclosure provides a composition for cell lysis and nucleic acid extraction, the composition comprising: a ribonuclease inhibitor; and a buffer.

[0028] According to one embodiment of the present disclosure, the composition for cell lysis and nucleic acid extraction enables the minimization of time required for nucleic acid amplification-based molecular diagnostic processes by omitting a separate process for purifying and eluting the cell-containing solution and using a polymerase chain reaction with the cell-lysed solution.

[0029] Referring to Figure 1, conventional molecular diagnostic methods typically include: obtaining a sample (step S10); extracting intracellular nucleic acids, such as DNA or RNA, from the sample (step S30); subjecting the sample to a lysis (step S31) process, an elution (step S33) process, and a purification (step S50) process; and adding additional PCR buffer to the eluted solution and performing a reverse transcription polymerase chain reaction (RT-PCR) (step S70) and PCR (step S90). The nucleic acids amplified by PCR are then used for diagnosis. However, the problem with conventional methods is that specialized equipment is required for the lysis, elution, and purification processes, and solutions or various consumables, such as various plates and / or tubes made of plastic, must be used continuously during these processes.

[0030] However, in conventional molecular diagnostic methods, the Ct value is often low because PCR is performed on samples with high concentrations after nucleic acid extraction and subsequent sample concentration. Conversely, the limitation of direct PCR (d-PCR) is that the Ct value may inevitably be higher than that in conventional molecular diagnostic methods because the concentration of the cell sample used for PCR is reduced by diluting the cell sample with lysis buffer. Therefore, there is a need for a molecular diagnostic method that can shorten the diagnostic time by minimizing RNA damage and loss and maximizing PCR efficiency even when extracting RNA from a small amount of cell sample.

[0031] Furthermore, when performing chemical lysis processes using surfactants in conventional d-PCR, the problem is that ribonuclease is inevitably contained within the sampled cells. That is, the problem is that during cell sampling, ribonuclease accompanies the cells, and during the lysis of cells using surfactants, the ribonuclease degrades the ribonucleic acid from the cells, leading to a rapid decrease in PCR efficiency.

[0032] According to one embodiment of this disclosure, the composition for cell lysis and nucleic acid extraction contains a ribonuclease inhibitor. Specifically, the composition contains a ribonuclease inhibitor capable of inhibiting ribonucleases that are not inactivated even upon heating, thereby simplifying molecular diagnostics and reducing the time required for molecular diagnostics.

[0033] According to one embodiment of this disclosure, the ribonuclease inhibitor may include an inhibitor of ribonuclease A. Since an inhibitor of ribonuclease A is selected as the ribonuclease inhibitor as described above, molecular diagnosis can be simplified and the time required for molecular diagnosis can be reduced by inactivating temperature-stable ribonuclease A while simultaneously inhibiting other ribonucleases by heating.

[0034] The characteristics of ribonuclease are shown in Table 1 below.

[0035] [Table 1] Ribonuclease A Ribonuclease T2 Ribonuclease T1 Ribonuclease H Ribonuclease P Ribonuclease I Necessary conditions for RNA division - - - Divalent metal ions Divalent metal ions - The principle of RNA division Specific splitting at the 3' end of unpaired cytosine / uracil (pyrimidine) residues in single-stranded ribonucleic acid (SsRNA) Splitting of all residues (preferably residue A) Splitting of unpaired G residues at the 3' end Hydrolysis of RNA phosphodiester bonds in DNA / RNA hybrids Hydrolysis of phosphodiester bonds in pre-tRNA Similar to T2 Splitting of all residues Temperature-related characteristics Stable at temperatures up to 100°C - 20℃ to 50℃ Inactivation was achieved by heat treatment at 60°C for 10 minutes. It is activated at 50°C and deactivated by heat treatment at 65°C for 10 minutes. Inactivation was achieved by heat treatment at 70°C for 20 minutes.

[0036] Specifically, ribonucleases correspond to temperature-dependent enzymes. However, when heated, ribonuclease T2, ribonuclease T1, ribonuclease H, ribonuclease P, and ribonuclease I are inactivated at low temperatures and lose their RNA degradation activity, but ribonuclease A remains stable even when heated to 100°C. Therefore, there is a problem that not all ribonucleases can be inactivated by heating.

[0037] Figure 2 illustrates a schematic diagram of a molecular diagnostic method according to an embodiment of the present disclosure, and a schematic diagram briefly illustrating the reactions between components in a tube.

[0038] Referring to Figures 2(a) and 2(b), cell or virus samples are obtained from the human body. A mixture is prepared by adding the obtained sample to a composition for cell lysis and nucleic acid extraction, wherein a ribonuclease A inhibitor contained in the mixture inactivates ribonuclease A in the sample. All ribonucleases except ribonuclease A are thermally inactivated by heating the mixture, and nucleic acids (i.e., RNA or DNA) in the cells are extracted by thermal lysis of the cells. Subsequently, when the extracted nucleic acids are mixed with primers, probes, and premixes and subjected to RT-PCR and PCR, the amplification time of the nucleic acids can be reduced.

[0039] According to one embodiment of this disclosure, the ribonuclease inhibitor may be derived from a protein. Specifically, the ribonuclease inhibitor may have a large molecular size. That is, the ribonuclease inhibitor may be derived from a protein and may have a large molecular size, and may bind itself to ribonuclease to form a large molecule, thereby preventing inhibition of the PCR reaction. More specifically, the ribonuclease inhibitor may be one selected from ribonuclease inhibitors derived from rat lungs, ribonuclease inhibitors derived from human placenta, and combinations thereof. The ribonuclease inhibitor derived from rat lungs may be a Nanohelix ribonuclease inhibitor (RI) (Nanohelix Co., Ltd.). The ribonuclease inhibitor may be selected from nano-helical Helix Ayme ribonuclease inhibitor (RNI2000), Themo Scientific RiboLock inhibitor (EO0381), Invitrogen RNaseOUT recombinant ribonuclease inhibitor (10777019), Takara recombinant ribonuclease inhibitor (2313A), Invitrogen™ SUPERase·In™ ribonuclease inhibitor (AM2694), Applied Biosystems™ ribonuclease inhibitor (N8080119), Roche Protector, etc. Protector ribonuclease inhibitors (RNAINH-RO / 3335399001), Sigma-Aldrich ribonuclease inhibitors for humans (R2520), Promega ribonuclease inhibitors (RNasin®) / RNasin® plus ribonuclease inhibitors, New England BioLabs Inc. ribonuclease inhibitors for mice (M0314), New England BioLabs Inc. ribonuclease inhibitors for humans, placenta (M0307), ABclonal Technology ribonuclease inhibitors for mammals (RK21401), BioVision RNaseOFF ribonuclease inhibitors (M1238), Biosystems RiboShield™ ribonuclease inhibitors (PB30).23-02), Blirt RIBOPROTECT Hu (RT35), highQu GmbH SecurRIN™ Advanced Ribonuclease Inhibitor (RNI0305), Enzynomics Ribonuclease Inhibitor (M007), Meridian Bioscience RiboSafe Ribonuclease Inhibitor (BIO-65027), QIAGEN Ribonuclease Inhibitor (Y9240L), Lucigen RiboGuard™ Ribonuclease Inhibitor (RG90925), Jena Bioscience Recombinant Ribonuclease Inhibitor (PCR392S), abm RNaseOFF Ribonuclease Inhibitor (G138), biotechrabbit Ribonuclease Inhibitor (BR0400901), BioFACT™ Ribonuclease Inhibitor (RI This refers to one of the following groups: 152-20h), Canvax ribonuclease inhibitor (P0269), ShineGene ribonuclease inhibitor (RNase inhibitor) (ZP00801), TOYOBO ribonuclease inhibitor (SIN-201), and combinations thereof. As mentioned above, protein-derived ribonuclease inhibitors are characterized by their large molecular size, and when using protein-derived ribonucleases with large molecular sizes, they can prevent inhibition of polymerase chain reaction (PCR) in the molecular diagnostic process described later. However, chemical-derived ribonuclease inhibitors (e.g., PVSA) have small molecular sizes and are therefore suitable for inhibiting polymerase chain reaction (PCR) in molecular diagnostic processes. Substances derived from chemicals (e.g., guanidinium isothiocyanate (GITC)) also inhibit ribonucleases, but are known to inhibit PCR reactions. Furthermore, reducing agents (e.g., β-mercaptoethanol) can be used as ribonuclease inhibitors, but these have drawbacks regarding long-term storage and safety. Therefore, since ribonuclease inhibitors are selected from protein-derived inhibitors as described above, they can prevent inhibition of polymerase chain reaction (PCR) during molecular diagnostics, thereby reducing the time required for the molecular diagnostic process.

[0040] According to one embodiment of this disclosure, the composition for cell lysis and nucleic acid extraction contains a buffer. Because the composition for cell lysis and nucleic acid extraction contains the buffer described above, it can improve the sensitivity of polymerase chain reaction (PCR) during molecular diagnostics, thereby reducing the time required for the molecular diagnostic process.

[0041] According to one embodiment of this disclosure, the pH of the buffer can be from 6.0 to 9.0. Specifically, the pH of the buffer can be 6.1 to 8.9, 6.2 to 8.7, 6.3 to 8.6, 6.4 to 8.5, 6.5 to 8.4, 6.6 to 8.3, 6.7 to 8.2, 6.8 to 8.1, 6.9 to 8.0, 7.0 to 7.9, 7.1 to 7.8, 7.2 to 7.7, 7.3 to 7.6, or 7.4 to 7.5. When the pH of the buffer is adjusted to the above range, the efficiency of the cell lysis process can be improved, and the reaction between the ribonuclease inhibitor and the ribonuclease can be promoted.

[0042] According to one embodiment of this disclosure, the buffer may contain any one selected from glycerol, hydroxyethylpiperazine ethanesulfonic acid (HEPES), dithiothreitol (DTT), potassium chloride, and combinations thereof. Since the components contained in the buffer are selected from the above-mentioned components, the efficiency of the cell lysis process can be improved, the reaction between the ribonuclease inhibitor and the ribonuclease can be promoted, and the sensitivity of polymerase chain reaction (PCR) in molecular diagnostics can be increased, thereby reducing the time required for the molecular diagnostic process.

[0043] Another embodiment of this disclosure provides a method for cell lysis and nucleic acid extraction, the method comprising: a step of preparing a mixture by adding a sample containing nucleic acid to a composition for cell lysis and nucleic acid extraction; a first heating step of maintaining the mixture at a temperature of 25°C to 45°C; and a second heating step of maintaining the heated mixture at a temperature of 75°C to below 100°C.

[0044] The method for cell lysis and nucleic acid extraction according to an embodiment of the present disclosure can improve the accuracy of molecular diagnostics by inactivating factors that inhibit the accuracy of polymer chain reactions through heating during the nucleic acid extraction process.

[0045] Figure 3 is a flowchart illustrating a molecular diagnostic method according to an embodiment of the present disclosure. The molecular diagnostic method may include the following steps: a sampling step of obtaining a biological sample from a human body; a step of preparing a mixture by adding the obtained sample to a composition for cell lysis and nucleic acid extraction (step S110); a first heating step of culturing the mixture (step S130); and a second heating step of thermally pyrolyzing the cultured mixture (step S150).

[0046] According to one embodiment of this disclosure, the molecular diagnostic method includes a step (step S110) of preparing a mixture by adding a nucleic acid-containing sample to a composition for cell lysis and nucleic acid extraction. Specifically, since a nucleic acid-containing sample (i.e., a biological sample containing ribonuclease obtained from a human body) is added to the composition for cell lysis and nucleic acid extraction, a ribonuclease inhibitor contained in the composition can be used to inactivate the ribonuclease, thereby protecting RNA and other components lysed from the cells from the effects of ribonuclease before performing the second heating (thermal lysis) step, which will be described later. Furthermore, since a specific ribonuclease inhibitor is used, PCR sensitivity can be improved and the time required for molecular diagnostics can be reduced by minimizing unnecessary components.

[0047] Where the details of the methods for cell lysis and nucleic acid extraction overlap with those of the components for cell lysis and nucleic acid extraction in this specification, they will not be repeated herein.

[0048] According to one embodiment of this disclosure, prior to the step of preparing the mixture (step S110), the method may further include a step of obtaining a biological sample from a human body. Specifically, the biological sample from a human body may be a cellular sample containing nucleic acids (i.e., DNA and / or RNA), such as blood, bodily fluids, or saliva, and may be obtained without restriction. Since the biological sample is obtained from a human body prior to performing the step of preparing the mixture (step S110) as described above, molecular diagnosis can be readily performed by amplifying a molecular diagnostic target (e.g., the SAR-CoV-2 gene that causes coronavirus disease (COVID) 19).

[0049] According to one embodiment of this disclosure, the method includes a first heating step (step S130) of maintaining the mixture at a temperature of 25°C to 45°C. Specifically, the first heating step may be a culturing step, in which the ribonuclease and ribonuclease inhibitor contained in the mixture are sufficiently bound together, thereby inactivating the ribonuclease. More specifically, the temperature of the first heating step may be 26°C to 44°C, 27°C to 43°C, 28°C to 42°C, 29°C to 41°C, 30°C to 40°C, 31°C to 39°C, 32°C to 38°C, 33°C to 37°C, or 34°C to 37°C. Preferably, the temperature of the first heating step may be 37°C. When the temperature of the first heating (culturing) step is controlled within the above range, the ribonuclease can be inactivated by promoting the reaction between the ribonuclease and the ribonuclease inhibitor, and the degradation of RNA released from the cell can be prevented by inactivating the ribonuclease before thermal lysis of the cell.

[0050] According to one embodiment of this disclosure, the method includes a second heating step (step S150) of maintaining the heated mixture at a temperature of 75°C to below 100°C. Specifically, the second heating step may be a thermal cell lysis step, i.e., exposing the DNA and / or RNA contained in the cells to the outside of the cells by lysing the cells in a sample containing nucleic acids contained in the mixture, wherein the sample is a cell-containing biological sample from a human body. Furthermore, this step can achieve thermal inactivation of ribonucleases while thermally lysing the cells, and can also simultaneously achieve additional inactivation of intracellular components that inhibit PCR. Specifically, the second heating step (step S150) can simultaneously achieve thermal lysis of cells, thermal inactivation of ribonucleases, and inactivation of intracellular components. Specifically, the second heating step can be a step of maintaining the heated mixture at a temperature of 76°C to 99°C, 77°C to 98°C, 76°C to 97°C, 77°C to 96°C, 78°C to 95°C, 79°C to 94°C, 80°C to 93°C, 81°C to 92°C, 82°C to 91°C, 83°C to 90°C, 84°C to 89°C, 85°C to 88°C, or 86°C to 87°C. Preferably, the second heating step can be a step of maintaining the heated mixture at a temperature of 94.5°C to 95.5°C or 95°C. Since the temperature of the second heating (thermal lysis) step is controlled within the above range, the separate additive for inhibiting ribonuclease can be eliminated, thereby reducing the concentration of substances other than nucleic acids and minimizing interfering factors. Furthermore, since no separate additive for inhibiting ribonuclease is contained, PCR inhibition caused by additives can be prevented. In addition, intracellular substances can be inactivated simultaneously with the inactivation of ribonucleases other than ribonuclease A, and intracellular nucleic acids (DNA and / or RNA) can be exposed by thermal lysis of cells, thereby reducing the time required for molecular diagnosis.

[0051] According to one embodiment of this disclosure, the method does not include separate elution and purification steps after extracting nucleic acids from a sample containing cells. Since, as described above, separate elution and purification steps are not performed after nucleic acid extraction, the time required for molecular diagnostics can be reduced, and the cost of molecular diagnostics can be lowered because no dedicated equipment or consumables for nucleic acid extraction are used.

[0052] According to one embodiment of this disclosure, the first heating step and the second heating step can each be performed for 1 minute to 30 minutes. Specifically, the first heating step and the second heating step can each be performed for 2 minutes to 29 minutes, 3 minutes to 28 minutes, 4 minutes to 27 minutes, 5 minutes to 26 minutes, 6 minutes to 25 minutes, 7 minutes to 24 minutes, 8 minutes to 23 minutes, 9 minutes to 22 minutes, 10 minutes to 21 minutes, 11 minutes to 20 minutes, 12 minutes to 19 minutes, 13 minutes to 18 minutes, 14 minutes to 17 minutes, or 15 minutes to 16 minutes. More specifically, the first heating step and the second heating step can each be performed for 4.5 minutes to 5.5 minutes, or 5 minutes. Since the time for each of the first heating step and the second heating step is controlled within the above ranges, the inactivation of ribonuclease can be maximized and the effect of thermal lysis of cells can be improved.

[0053] According to one embodiment of this disclosure, based on a 30 μL volume of mixture, the concentration of ribonuclease inhibitor in the mixture can be from 7.5 units / reaction to 60.0 units / reaction. The concentration of ribonuclease inhibitor in the mixture can vary with the increase or decrease of the total volume of the mixture. Specifically, the concentration of ribonuclease inhibitor in the mixture can be from 7.5 units / reaction to 60.0 units / reaction, 8.0 units / reaction to 59.0 units / reaction, 9.0 units / reaction to 58.0 units / reaction, 10.0 units / reaction to 57.0 units / reaction, 15.0 units / reaction to 55.0 units / reaction, 20.0 units / reaction to 50.0 units / reaction, 25.0 units / reaction to 45.0 units / reaction, or 30.0 units / reaction to 40.0 units / reaction. More specifically, the concentration of the ribonuclease inhibitor in the mixture can be from 7.5 units / reaction to 52.5 units / reaction, 30.0 units / reaction to 52.5 units / reaction, or 30.0 units / reaction to 45.0 units / reaction. When the concentration of the ribonuclease inhibitor in the mixture is controlled within the above range, the inactivation of ribonuclease can be maximized before thermal lysis of cells, and the degradation of RNA released from cells after thermal lysis can be prevented, and factors inhibiting subsequent PCR can be minimized, thereby reducing the time required for molecular diagnostics.

[0054] As used herein, the term "unit / reaction (U / rxn)" refers to the amount of ribonuclease inhibitor required to inhibit 50% of the activity of 5 nanograms (ng) of ribonuclease A per reaction.

[0055] Another embodiment of this disclosure provides a molecular diagnostic method, the molecular diagnostic method comprising the following steps: adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by a method for cell lysis and nucleic acid extraction; and amplifying the extracted nucleic acids by polymerase chain reaction.

[0056] A molecular diagnostic method according to an embodiment of the present disclosure can reduce the cost of molecular diagnostics by minimizing the use of specialized equipment and consumables for extraction.

[0057] Referring to FIG3, a method according to an embodiment of the present disclosure includes the step of adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by a method for cell lysis and nucleic acid extraction (step S170). As used herein, the phrase "a composition containing a solution (containing primers and probes) and a premix" may refer to a PCR sample. Specifically, when a PCR sample containing a solution (containing primers and probes) and a premix is ​​added to a mixture containing nucleic acids extracted by a method for cell lysis and nucleic acid extraction, the components for nucleic acid amplification are fully provided, and nucleic acid amplification is readily achieved.

[0058] According to one embodiment of this disclosure, the PCR sample contains primers, and the nucleotide sequences of the primers used in this disclosure are not particularly limited, but may be the 2019-COVID primer sequences (N1) published by the Centers for Disease Control and Prevention (CDC). These sequences may be those published at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf, and any primers may be used without restriction, as long as they are used for PCR.

[0059] According to one embodiment of this disclosure, the PCR sample contains a probe, and the nucleotide sequence of the probe used in this disclosure may be the 2019-COVID probe sequence (N1) published by the Centers for Disease Control and Prevention (CDC). The sequence may be the one published at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf, and any probe may be used without restriction, as long as it is used for PCR.

[0060] According to one embodiment of this disclosure, the PCR sample contains a premix, and the premix used in this disclosure is not particularly limited, but the RealHelix™ qRT-PCR kit [v6] (UDG system; NanoHelix Ltd.) is preferred. Any premix can be used without limitation, as long as it is used for PCR.

[0061] According to one embodiment of this disclosure, the step of adding the solution and premix can be a step of adding a PCR sample containing a solution (containing primers and probes) and a premix to a well (or tube) containing a mixture containing nucleic acids extracted by a method for cell lysis and nucleic acid extraction. When the sample is removed from the mixture and placed in a separate tube as described above, without adding PCR sample, all the extracted nucleic acids can be used for nucleic acid amplification, thereby minimizing the loss of target genes and maximizing PCR efficiency. Furthermore, since a separate solution transfer process is not required, PCR preparation time can be reduced. In addition, the use of nucleic acids exposed by thermal lysis of cells can be maximized, and concentration dilution due to the addition of PCR sample can be prevented, thereby avoiding the time required for molecular diagnostics and the inhibition of PCR by additives.

[0062] According to one embodiment of this disclosure, the molecular diagnostic method includes a step of amplifying extracted nucleic acids by polymerase chain reaction (step S190). Specifically, in the step of amplifying extracted nucleic acids by polymerase chain reaction, RT-PCR (step S191) and PCR (step S193) may be performed sequentially. Since the extracted nucleic acids are amplified by polymerase chain reaction, nucleic acids for molecular diagnosis can be obtained and the time required for molecular diagnosis can be minimized.

[0063] According to one embodiment of this disclosure, in a molecular diagnostic method, nucleic acids in wells (or tubes) containing a solution (containing primers and probes) and a premix can be amplified by polymerase chain reaction without separate purification. Since the nucleic acids in wells containing PCR samples are amplified by polymerase chain reaction as described above without separate purification, the time required for molecular diagnostics can be minimized.

[0064] Another embodiment of this disclosure provides the use of a composition for cell lysis and nucleic acid extraction, the composition comprising: a ribonuclease inhibitor; and a buffer solution.

[0065] Another embodiment of this disclosure provides a kit for cell lysis and nucleic acid extraction or molecular diagnostics, the kit comprising: a ribonuclease inhibitor; and a buffer solution.

[0066] Another embodiment of this disclosure provides the use of a composition for preparing a kit for nucleic acid extraction or molecular diagnostics, the composition comprising a ribonuclease inhibitor and a buffer solution.

[0067] According to one embodiment of this disclosure, by using a composition comprising a ribonuclease inhibitor and a buffer solution, the purification and elution processes of the solution are omitted, and a polymerase chain reaction is performed using the solution, thereby minimizing the time required for nucleic acid amplification and molecular diagnostics. Therefore, it can be used for cell lysis and nucleic acid extraction, as a kit for cell lysis and nucleic acid extraction, for molecular diagnostics, and for the manufacture of such products.

[0068] The cell lysis and nucleic acid extraction uses, kits and uses for preparing kits, components for cell lysis and nucleic acid extraction, molecular diagnostics, ribonuclease inhibitors and buffer solutions of the present invention described in the above embodiments are as described above.

[0069] In the following description, this disclosure will be illustrated in detail with reference to examples. However, examples based on this disclosure can be modified in various different forms, and the scope of this disclosure is not to be construed as limited to the examples described below. The examples provided in this specification are intended to explain this disclosure more fully to those skilled in the art.

[0070] <Compounds used in Examples 1 to 4 and conditions for PCR>

[0071] The samples obtained in Examples 1 to 4 below were samples stored in viral transport media after being sampled using clinical swabs, and purified target RNA was used as an additional RNA sample.

[0072] In addition, a ribonuclease inhibitor derived from rat lung (Nanospiral Ltd.) was used as the ribonuclease inhibitor in Examples 1 to 4 below, and a mixture of glycerol, hydroxyethylpiperazine ethanesulfonic acid (HEPES), dithiothreitol (DTT) and potassium chloride was used as a buffer.

[0073] Furthermore, as probes for the PCR samples added for PCR in Examples 1 to 4 below, the 2019-COVID probe sequence (N1) published by the Centers for Disease Control and Prevention (CDC) was used. The 2019-COVID probe sequence (N1) is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. As primers, the 2019-COVID primer sequence (N1) published by the Centers for Disease Control and Prevention (CDC) was used. The 2019-COVID primer sequence (N1) is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. As a premix, the RealHelix™ qRT-PCR kit [v6] (UDG system, Nanospiral Ltd.) was used.

[0074] In Examples 1 to 4 below, RT-PCR and PCR were performed under the following conditions: (1) at 50°C for 10 minutes; (2) at 95°C for 5 minutes, and then (3) 40 cycles were performed, each cycle consisting of 10 seconds at 95°C and 30 seconds at 58°C. During this process, nucleic acids were amplified, and the Ct (critical cycle) value was measured, which is the minimum threshold value at which the amplification result can be confirmed.

[0075] Example 1 (Evaluation of the effect of pyrolysis)

[0076] Figure 4 is a schematic diagram illustrating the molecular diagnostic method according to Example 1, and a graph showing the Ct values ​​obtained in Examples 1-1 and 1-2.

[0077] Specifically, Figure 4(a) is a schematic diagram illustrating the molecular diagnostic method according to Example 1. Referring to Figure 4(a), in Example 1-1, a sample containing nucleic acid was obtained, and distilled water was added to the obtained sample, followed by thermal lysis at 95°C for 5 minutes. Next, prior to RT-PCR, a separately cultured RNA sample was added, and then RT-PCR and PCR were performed sequentially.

[0078] Example 1-2 was performed in the same manner as in Example 1-1, except that thermal decomposition was not performed.

[0079] Figure 4(b) is a graph showing the Ct values ​​obtained in Examples 1-1 and 1-2. Referring to Figure 4(b), it is confirmed that the Ct value of Example 1-1 is 0.5 lower than that of Example 1-2, which indicates that the PCR inhibitors (excluding ribonuclease A inhibitors) accompanying the samples were inactivated during the thermal lysis process.

[0080] Example 2 (Evaluation of the effectiveness of ribonuclease inhibitors based on their type)

[0081] Figure 5 is a schematic diagram illustrating the molecular diagnostic method according to Example 2, and a graph showing the Ct values ​​obtained in Examples 2-1 to 2-4.

[0082] Specifically, Figure 5(a) is a schematic diagram illustrating the molecular diagnostic method according to Example 2. Referring to Figure 5(a), in Example 2-1, a sample containing nucleic acid was obtained, and distilled water was added to the obtained sample, followed by thermal lysis at 95°C for 5 minutes. Next, prior to RT-PCR, a separately cultured RNA sample was added, and then RT-PCR and PCR were performed sequentially.

[0083] Example 2-2 was performed in the same manner as in Example 2-1, except that the RNA sample was added together with the sample to distilled water.

[0084] Example 2-3 was performed in the same manner as in Example 2-2, except that distilled water containing a ribonuclease inhibitor was used instead of distilled water.

[0085] Example 2-4 was carried out in the same manner as in Example 2-2, except that distilled water containing polyvinylsulfonic acid (PVSA) (which is a ribonuclease inhibitor derived from chemicals) was used instead of distilled water.

[0086] Figure 5(b) is a graph showing the Ct values ​​obtained in Examples 2-1 to 2-4. Referring to Figure 5(b), it is confirmed that in Example 2-1, most of the ribonuclease contained in the sample was inactivated by thermal lysis, and only ribonuclease A remained active. Therefore, the RNA sample added immediately before RT-PCR was amplified and a low Ct value was obtained. In contrast, it is confirmed that in Example 2-2, the Ct value increased because the ribonuclease contained in the sample had some activity before thermal lysis, and even under the same conditions as in Example 2-1, only the order of addition was changed, but a Ct value approximately 4.8 to 8 higher than that in Example 2-1 was obtained. Furthermore, it is confirmed that in the case of Example 2-3, ribonuclease A was inactivated by adding a ribonuclease inhibitor along with the sample, and therefore a Ct value approximately 2.6 lower than that in Example 2-2 was obtained. However, in Examples 2-4, where chemically derived ribonuclease inhibitors were used instead of protein-derived ribonuclease inhibitors, the Ct values ​​obtained were 0.02 higher than those in Example 2-2 due to the PCR inhibition effect of the ribonuclease inhibitors.

[0087] Example 3 (Evaluating the effectiveness of buffers based on their type)

[0088] Figure 6 is a schematic diagram illustrating the molecular diagnostic method according to Example 3, and a graph showing the Ct values ​​obtained in Example 3-1 and Example 3-2.

[0089] Figure 6(a) is a schematic diagram illustrating the molecular diagnostic method according to Example 3. Referring to Figure 6(a), in Example 3-1, a sample containing nucleic acid was obtained, and distilled water and separately cultured RNA samples were added to the obtained sample, followed by thermal lysis at 95°C for 5 minutes. Then, RT-PCR and PCR were performed sequentially.

[0090] Example 3-2 was performed in the same manner as in Example 3-1, except that a buffer was used instead of distilled water.

[0091] Figure 6(b) is a graph showing the Ct values ​​obtained in Examples 3-1 and 3-2. Referring to Figure 6(b), it is confirmed that the Ct value obtained in Example 3-2 is 1.8 lower than the Ct value in Example 3-1, which indicates that even if only the buffer is changed, the PCR amplification effect is improved.

[0092] Example 4 (Evaluation of its effectiveness based on the type of thermal lysis, buffer, and ribonuclease inhibitor)

[0093] Figure 7 is a schematic diagram illustrating the molecular diagnostic method according to Example 4, and a graph showing the Ct values ​​obtained in Example 4-1 and Example 4-2.

[0094] Specifically, Figure 7(a) is a schematic diagram illustrating the molecular diagnostic method according to Example 4. Referring to Figure 7(a), in Example 4-1, a sample containing nucleic acid was obtained, and the obtained sample was added to distilled water and separately cultured RNA sample, followed by thermal lysis at 95°C for 5 minutes. Then, RT-PCR and PCR were performed sequentially.

[0095] In Example 4-2, a sample containing nucleic acid was obtained and added to distilled water, followed by thermal lysis at 95°C for 5 minutes. Next, prior to RT-PCR, a separately cultured RNA sample was added. Then, RT-PCR and PCR were performed sequentially.

[0096] Figure 7(b) is a graph showing the Ct values ​​obtained in Examples 4-1 and 4-2.

[0097] Referring to Figure 7(b), it was confirmed that a high Ct value was obtained in Example 4-1 because the concentration of RNA used for nucleic acid amplification was reduced due to the degradation of the RNA sample by the ribonuclease present with the sample. In contrast, it was confirmed that a low Ct value of 4.7 was obtained in Example 4-2, in which RNA sample was added immediately before RT-PCR for nucleic acid amplification, because a very small amount of RNA was inactivated and a high concentration of RNA was present.

[0098] Figure 8 is a schematic diagram illustrating the molecular diagnostic methods according to Examples 4-3 to 4-6. Referring to Figure 8, Example 4-3 was performed in the same manner as in Example 4-1, except that a thermal pyrolysis process was added.

[0099] Example 4-4 was performed in the same manner as in Example 4-3, except that a ribonuclease inhibitor was added to distilled water.

[0100] Example 4-5 was performed in the same manner as in Example 4-3, except that a buffer was added to the distilled water.

[0101] Except for the addition of ribonuclease inhibitors and buffers to distilled water, Examples 4-6 were carried out in the same manner as in Examples 4-3.

[0102] It was confirmed that the Ct value obtained in Example 4-3 was 0.5 lower than the Ct value obtained in Example 4-1 because the ribonuclease contained in the sample was thermally inactivated during the thermal lysis process.

[0103] Furthermore, it was confirmed that the Ct value obtained in Example 4-4 was 3.1 lower than the Ct value obtained in Example 4-1, because it exhibited the heat inactivation effect confirmed in Example 4-3, and the ribonuclease inhibitor minimized RNA degradation by removing ribonuclease A.

[0104] Furthermore, it was confirmed that the Ct values ​​obtained in Examples 4-5 were 1.8 lower than the Ct values ​​obtained in Example 4-1, indicating that the buffer improved the PCR amplification effect.

[0105] Furthermore, it was confirmed that the Ct values ​​obtained in Examples 4-6 were 4.9 lower than the Ct values ​​obtained in Example 4-1. This indicates that the heat inactivation effect confirmed in Example 4-3 and the inactivation effect of the ribonuclease inhibitor on ribonuclease A confirmed in Example 4-4 were demonstrated, and at the same time, the buffer removal effect of the PCR inhibitor as confirmed in Example 4-5 was also demonstrated, thereby obtaining Ct values ​​comparable to those in Example 4-2.

[0106] <Preparation Example>

[0107] The sample obtained in this preparation example was a sample stored in a viral transport medium after clinical swab sampling.

[0108] Furthermore, a ribonuclease inhibitor derived from rat lung (Nanospiral Inc.) was used as the ribonuclease inhibitor in this preparation example, and a mixture of glycerol, hydroxyethylpiperazine ethanesulfonic acid (HEPES), dithiothreitol (DTT), and potassium chloride was used as a buffer. In addition, a mixture of the obtained sample, the ribonuclease inhibitor, and the buffer was prepared.

[0109] Furthermore, as probes for the PCR sample added for PCR in this preparation example, the 2019-COVID probe sequence (N1) released by the Centers for Disease Control and Prevention (CDC) was used. The 2019-COVID probe sequence (N1) is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. As primers, the 2019-COVID primer sequence (N1) released by the Centers for Disease Control and Prevention (CDC) was used. The 2019-COVID primer sequence (N1) is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. As a premix, the RealHelix™ qRT-PCR kit [v6] (UDG system, Nanospiral Ltd.) was used.

[0110] In this preparation example, RT-PCR and PCR were performed under the following conditions: (1) at 50°C for 10 minutes; (2) at 95°C for 5 minutes, and then (3) 40 cycles were performed, each cycle consisting of 10 seconds at 95°C and 30 seconds at 58°C. During this process, nucleic acids were amplified, and the Ct (critical cycle) value was measured, which is the minimum threshold value at which the amplification result can be confirmed.

[0111] Experimental Example 1 (Comparison with conventional PCR including extraction process)

[0112] Figure 9 is a graph showing the Ct values ​​obtained in a conventional PCR method including RNA extraction and purification processes and in the preparation example.

[0113] In the preparation example, a sample containing nucleic acid was obtained, and distilled water, ribonuclease inhibitor, and buffer were added to the obtained sample. Then, thermal lysis was performed at 95°C for 5 minutes. Then, RT-PCR and PCR were performed sequentially.

[0114] Figure 9 shows the results of comparing the PCR method performed according to the conventional technique shown in Figure 1 with the preparation example. Referring to Figure 9, it is confirmed that when PCR is performed after thermal lysis following the addition of a buffer containing a ribonuclease inhibitor to the sample, a Ct value comparable to that of the conventional PCR method is obtained.

[0115] Experimental Example 2 (Evaluation of the effect based on the concentration of ribonuclease inhibitor in the mixture during the first heating step)

[0116] Figure 10 is a graph showing the change of Ct value with the concentration of ribonuclease inhibitor in the first heating step (culturing) in the preparation example.

[0117] Specifically, in the preparation example, before performing RT-PCR and PCR, the mixture of the preparation example was subjected to a first heating step (incubation) at 37°C for 5 minutes while changing the concentration of the ribonuclease inhibitor in the mixture, and the Ct value of each concentration was measured. More specifically, in the preparation example, a sample containing nucleic acid was obtained, and the obtained sample was added to distilled water, ribonuclease inhibitor, and buffer, and then thermally lysed at 95°C for 5 minutes. Subsequently, the mixture of the preparation example was subjected to a first heating step (incubation) at 37°C for 5 minutes while changing the concentration of the ribonuclease inhibitor in the mixture, and then RT-PCR and PCR were performed sequentially. The changed concentrations were 0 units / reaction (U / rxn), 7.5 units / reaction, 15 units / reaction, 22.5 units / reaction, 30 units / reaction, 37.5 units / reaction, 45 units / reaction, and 52.5 units / reaction, and the Ct value of each concentration was measured.

[0118] Referring to Figure 10, it was confirmed that a high Ct value was obtained at 0 units / reaction due to the absence of ribonuclease inhibitors. Subsequently, it was confirmed that the Ct value gradually decreased with increasing ribonuclease inhibitor concentration from 7.5 units / reaction to 45 units / reaction. However, it was confirmed that at 52.5 units / reaction, the Ct value increased despite the increase in ribonuclease inhibitor concentration, but it was lower than the Ct value at the concentration of 7.5 units / reaction.

[0119] Experiment 3 (Evaluating the effect based on the temperature of the first heating step)

[0120] Figure 11 is a graph showing the change of Ct value with the temperature of the first heating step in the preparation example.

[0121] Specifically, in the preparation example, before performing RT-PCR and PCR, the concentration of the ribonuclease inhibitor in the mixture of the preparation example was fixed at 30 units / reaction, and the mixture was subjected to a first heating step (incubation) for 5 minutes while changing the temperature of the first heating step, and the Ct value at each temperature was measured. More specifically, in the preparation example, a sample containing nucleic acid was obtained, and the obtained sample was added to distilled water, ribonuclease inhibitor, and buffer, and then subjected to thermal lysis at 95°C for 5 minutes. Thereafter, the concentration of the ribonuclease inhibitor in the mixture of the preparation example was fixed at 30 units / reaction, and the mixture was subjected to a first heating step (incubation) for 5 minutes while changing the temperature of the first heating step. Then, RT-PCR and PCR were performed sequentially. The varying temperatures were 25°C, 37°C, 45°C, and 60°C, and the Ct value at each temperature was measured.

[0122] Referring to Figure 11, it was confirmed that the Ct value was constant at temperatures ranging from 25°C to 37°C. Then, it was confirmed that the Ct value increased at temperatures of 45°C or higher, indicating that ribonuclease inhibitors are inhibited at temperatures of 45°C or higher.

[0123] Therefore, the composition for cell lysis and nucleic acid extraction according to an embodiment of the present disclosure, the nucleic acid extraction method using the composition, and the molecular diagnostic method using the composition can inactivate ribonuclease by using a composition containing a ribonuclease inhibitor as the composition for nucleic acid extraction and simultaneously heating it. This eliminates the need for a separate nucleic acid purification process and shortens the total experimental time. Furthermore, it can improve PCR efficiency by minimizing damage to RNA.

[0124] Although the present disclosure has been described above with reference to limited embodiments, the present disclosure is not limited thereto. It should be understood that those skilled in the art can make various changes and modifications to the present disclosure without departing from the technical spirit of the present disclosure and the equivalent scope of the appended claims. [Simplified Explanation of the Diagram]

[0023] Figure 1 is a flowchart illustrating a method for molecular diagnostics using polymerase chain reaction according to conventional techniques. Figure 2 illustrates a schematic diagram of a molecular diagnostic method according to an embodiment of the present disclosure, and a schematic diagram briefly illustrating the reactions between components in a tube. Figure 3 is a flowchart illustrating a molecular diagnostic method according to an embodiment of the present disclosure. Figure 4 is a schematic diagram illustrating a molecular diagnostic method according to Example 1, and a graph showing the Ct values ​​obtained in Examples 1-1 and 1-2. Figure 5 is a schematic diagram illustrating a molecular diagnostic method according to Example 2, and a graph showing the Ct values ​​obtained in Examples 2-1 to 2-4. Figure 6 is a schematic diagram illustrating a molecular diagnostic method according to Example 3, and a graph showing the Ct values ​​obtained in Examples 3-1 and 3-2. Figure 7 is a schematic diagram illustrating a molecular diagnostic method according to Example 4, and a graph showing the Ct values ​​obtained in Examples 4-1 and 4-2. Figure 8 is a schematic diagram illustrating molecular diagnostic methods according to Examples 4-3 to 4-6. Figure 9 is a graph showing the Ct values ​​obtained in the conventional polymerase chain reaction (PCR) method, including RNA extraction and purification processes, and in the preparation example. Figure 10 is a graph showing the Ct value as a function of the concentration of the ribonuclease inhibitor during the first heating step in the preparation example. Figure 11 is a graph showing the Ct value as a function of the temperature during the first heating step in the preparation example.

Claims

1. A composition for cell lysis and nucleic acid extraction, comprising: Ribonuclease inhibitors; And a buffer, wherein the ribonuclease inhibitor is derived from a protein, wherein the ribonuclease inhibitor includes an inhibitor of ribonuclease A, wherein based on a PCR reaction mixture of a total volume of 30 μL, the PCR reaction mixture comprises the composition, a sample containing nucleic acid, a premix, and a solution containing primers and probes, wherein the concentration of the ribonuclease inhibitor in the composition is 30.0 units / reaction to 45.0 units / reaction.

2. The composition for cell lysis and nucleic acid extraction as described in claim 1, wherein the buffer has a pH of 6.0 to 9.

0.

3. The composition for cell lysis and nucleic acid extraction as claimed in claim 1, wherein the buffer contains any one selected from glycerol, hydroxyethylpiperazine ethanesulfonic acid (HEPES), dithiothreitol (DTT), potassium chloride, and combinations thereof.

4. A method for cell lysis and nucleic acid extraction, comprising: The step of preparing a mixture by adding a sample containing nucleic acid to a composition for cell lysis and nucleic acid extraction as described in claim 1; A first heating step involves maintaining the mixture at a temperature of 25°C to 45°C without purification and elution processes; and a second heating step involves maintaining the heated mixture at a temperature of 75°C to below 100°C, wherein the first heating step and the second heating step are each performed for 1 minute to 30 minutes, wherein the PCR reaction mixture comprises the composition, a sample containing nucleic acids, a premix, and a solution containing primers and probes, based on a total volume of 30 μL, and the concentration of ribonuclease inhibitor in the mixture is 30.0 units / reaction to 45.0 units / reaction.

5. A molecular diagnostic method, comprising: The solution and premix containing primers and probes are added to a mixture containing nucleic acids extracted by the method for cell lysis and nucleic acid extraction as described in claim 4; And the extracted nucleic acids are amplified by polymerase chain reaction.

6. A kit for cell lysis and nucleic acid extraction or molecular diagnostics, comprising: The composition as described in claim 1.

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