Composition for cell lysis and nucleic acid extraction, nucleic acid extraction method using the same, and molecular diagnostic method using the same

Abstract

The present invention relates to a composition for cell lysis and nucleic acid extraction, a method for extracting nucleic acid using the same, and a method for molecular diagnosis using the same. More specifically, the present invention relates to a composition for cell lysis and nucleic acid extraction, a method for extracting nucleic acid using the same, and a method for molecular diagnosis using the same, which use a composition containing an RNase inhibitor as a solution for extracting nucleic acid, and perform a polymerase chain reaction without a separate purification process and elution process, including a step of heating at a specific temperature, thereby minimizing the time required for molecular diagnosis and minimizing the dedicated equipment and consumables used for extraction, thereby reducing the cost of molecular diagnosis.

Description

[Technical Field]

[0001] This specification claims the benefit of Korean Patent Application No. 10-2021-0101536, filed with the Korean Intellectual Property Office on August 2, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a composition for cell lysis and nucleic acid extraction, a nucleic acid extraction method using the same, and a molecular diagnostic method using the same. Specifically, the present invention relates to a composition for cell lysis and nucleic acid extraction, a nucleic acid extraction method using the same, and a molecular diagnostic method using the same, which use a composition containing an RNase inhibitor as a solution for nucleic acid extraction, and which performs a polymerase chain reaction (PCR) without a separate purification and elution process, including a heating step at a specific temperature, thereby minimizing the time required for molecular diagnosis and reducing the cost of molecular diagnosis by minimizing the amount of dedicated equipment and consumables used for extraction. [Background technology]

[0003] Recently, there has been a gradual increase in the demand for biological sample manipulation and biochemical analysis in order to cure or prevent human diseases by interpreting the causes of diseases at the genetic level based on the results of human genome research. In addition to disease diagnosis, there is also a demand for techniques to extract and analyze nucleic acids from biological samples or samples containing cells in a variety of fields, such as new drug development, preliminary testing for viral or bacterial infections, and forensic medicine.

[0004] On the other hand, molecular diagnosis typically involves extracting nucleic acids, which are DNA or RNA that contain genetic information, from the saliva or blood of a person infected with a virus or bacteria, and amplifying them to confirm whether or not the person is infected with the disease.

[0005] Figure 1 is a flowchart showing a conventional PPCR. Referring to Figure 1, conventional methods involve extracting nucleic acids (S30) containing genetic information from a sample (S10), amplifying the nucleic acids (S70, S90) through PPCR, and analyzing the results. Extracting nucleic acids requires the steps of lysis (S31), elution (S33), and purification (S50). However, the lysis and purification steps for nucleic acid extraction require specialized extraction equipment and consumable materials (e.g., plastic tools, magnetic beads, or solutions).

[0006] When nucleic acids are extracted using the above-mentioned conventional technologies, high-purity nucleic acids can be extracted, but the extraction process takes a long time, making it unsuitable for diagnosis or screening in emergency situations or emergency rooms. Furthermore, there are problems with applying this technology in situations where national quarantine is necessary due to the rapid spread of a virus, and with the need for continuous use of dedicated extraction equipment and consumables, which results in high costs for diagnosis.

[0007] Therefore, in order to solve the above problems, there is an urgent need to develop a technology that can perform PPCR by simply lysing cells without any purification or elution process by using a specific composition. Summary of the Invention [Problem to be solved by the invention]

[0008] The technical problem to be solved by the present invention is to provide a composition for cell lysis and nucleic acid extraction, which can perform a polymerase chain reaction using a cell lysing solution by heating a mixture containing the lysed cells using a composition containing specific components during the process of lysing cells to extract intracellular nucleic acids, thereby eliminating the need for separate purification and elution processes of the cell lysing solution, and a molecular diagnostic method using the same.

[0009] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] One embodiment of the present invention provides a composition for cell lysis and nucleic acid extraction, comprising an RNase inhibitor and a buffer solution.

[0011] According to one embodiment of the present invention, the RNase inhibitor may include an inhibitor that inhibits RNase A.

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

[0013] According to one embodiment of the present invention, the buffer solution may have a pH of 6.0 or more and a pH of 9.0 or less.

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

[0015] One embodiment of the present invention provides a method for cell lysis and nucleic acid extraction, comprising the steps of: adding a sample containing nucleic acids to the composition for cell lysis and nucleic acid extraction to prepare a mixture; performing a primary heating step to maintain the mixture at a temperature of 25°C or higher and 45°C or lower; and performing a secondary heating step to maintain the primarily heated mixture at a temperature of 75°C or higher and lower than 100°C.

[0016] According to one embodiment of the present invention, the first heating step and the second heating step may each be performed for 1 minute or more and 30 minutes or less.

[0017] According to one embodiment of the present invention, the concentration of the RNase inhibitor in the mixture may be 7.5 Units / reaction or more and 60.0 Units / reaction or less, based on a volume of the mixture of 30 μL.

[0018] One embodiment of the present invention provides a molecular diagnostic method comprising the steps of adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by the cell lysis and nucleic acid extraction method, and amplifying the extracted nucleic acids by polymerase chain reaction. [Effects of the Invention]

[0019] A composition for cell lysis and nucleic acid extraction according to one embodiment of the present invention can minimize the time required for the process of amplifying nucleic acids for molecular diagnosis by performing a polymerase chain reaction using the lysed cell solution, without the need for a separate purification and elution process of the lysed cell solution.

[0020] The cell lysis and nucleic acid extraction method according to one embodiment of the present invention can improve the accuracy of molecular diagnostics by inactivating factors that inhibit the accuracy of polymerization chain reaction through heating during the nucleic acid extraction process.

[0021] A molecular diagnostic method according to one embodiment of the present invention can reduce the cost of molecular diagnostics by minimizing the dedicated equipment and consumables used for extraction.

[0022] The effects of the present invention are not limited to those described above, and effects not mentioned herein will be clearly understood by those skilled in the art from the present specification and the accompanying drawings. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a flow chart showing a polymerase chain reaction according to the prior art. [Figure 2] FIG. 2 is a schematic diagram of a molecular diagnostic method according to one embodiment of the present invention and a simplified schematic diagram showing the reaction of components inside the tube. [Figure 3] FIG. 3 is a flowchart of a molecular diagnostic method according to one embodiment of the present invention. [Figure 4] FIG. 4 is a schematic diagram of the molecular diagnostic method according to Example 1 and a graph showing the Ct values ​​of Examples 1-1 and 1-2. [Figure 5] FIG. 5 is a schematic diagram of the molecular diagnostic method according to Example 2 and a graph showing the Ct values ​​of Examples 2-1 to 2-4. [Figure 6] FIG. 6 is a schematic diagram of the molecular diagnostic method according to Example 3 and a graph showing the Ct values ​​of Examples 3-1 and 3-2. [Figure 7] FIG. 7 is a schematic diagram of the molecular diagnostic method according to Example 4 and a graph showing the Ct values ​​of Examples 4-1 and 4-2. [Figure 8] FIG. 8 is a schematic diagram of the molecular diagnostic method according to Examples 4-3 to 4-6. [Figure 9] FIG. 9 is a graph showing the Ct values ​​of PCR including the RNA extraction and purification steps according to the prior art and the production example. [Figure 10] FIG. 10 is a graph showing the Ct value depending on the concentration of RNase inhibitor in the first heating step of a preparation example. [Figure 11] FIG. 11 is a graph showing the Ct value as a function of the temperature in the first heating step in the production examples. DETAILED DESCRIPTION OF THE INVENTION

[0024] Throughout this specification, when a part is described as "comprising" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless specifically stated to the contrary.

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

[0026] The present invention will be described in further detail below.

[0027] One embodiment of the present invention provides a composition for cell lysis and nucleic acid extraction, comprising an RNase inhibitor and a buffer solution.

[0028] The composition for cell lysis and nucleic acid extraction according to one embodiment of the present invention can minimize the time required for the process of amplifying nucleic acids for molecular diagnosis by performing a polymerase chain reaction using the lysed cell solution, without the need for a separate purification and elution process of the lysed cell solution.

[0029] Referring to FIG. 1, conventional molecular diagnostic methods involve collecting a sample (S10) to extract nucleic acids, such as intracellular DNA or RNA (S30), followed by lysis (S31), elution (S33), and purification (S50). An additional PCR buffer is added to the eluted solution to perform reverse transcription PCR (RT-PCR, S70) and polymerase chain reaction (PCR, S90). The nucleic acids amplified by PCR are then typically used for diagnosis. However, these methods have drawbacks, such as the need for dedicated equipment for the lysis, elution, and purification processes, and the need for continuous use of various consumables, such as solutions or plasticware plates and / or tubes, used in these processes.

[0030] However, in conventional molecular diagnostic methods, after nucleic acid extraction, the sample is concentrated and PCR is performed on a highly concentrated sample, which tends to result in low Ct values. In contrast, in direct PCR (d-PCR), cells are collected and diluted with a lysis buffer, which reduces the concentration of the sample used for PCR, resulting in a limitation that the Ct values ​​can only be higher than those of conventional molecular diagnostic methods. Therefore, there is a need for a molecular diagnostic method that can minimize RNA damage and loss, maximize PCR efficiency, and shorten PCR time, even when a small amount of cell sample is collected and RNA is extracted.

[0031] Furthermore, when chemically lysing cells using surfactants in conventional d-PCR, RNase is inevitably present in the collected cells. That is, RNase is present during the cell collection process, and when the cells are lysed using surfactants, the RNase degrades the RNA released from the cells, resulting in a rapid decrease in PCR efficiency.

[0032] According to one embodiment of the present invention, the composition for cell lysis and nucleic acid extraction contains an RNase inhibitor, specifically, an RNase inhibitor that can inhibit RNases that are not inactivated by heating, thereby simplifying molecular diagnostics and shortening the time required for molecular diagnostics.

[0033] According to one embodiment of the present invention, the RNase inhibitor may include an inhibitor that inhibits RNase A. As described above, by selecting the RNase inhibitor as an inhibitor that inhibits RNase A, other RNases can be inhibited by heating, and RNase A, which is stable to temperature, can be inactivated, thereby simplifying molecular diagnosis and shortening the time required for molecular diagnosis. The properties of RNA-degrading enzymes are as shown in Table 1 below.

[0034] [Table 1]

[0035] Specifically, RNases are enzymes that are affected by temperature. However, when heated, RNase T2, RNase T1, RNase H, RNase P, and RNase I become inactive at low temperatures and lose their RNA degradation activity, but RNase A remains stable even when heated up to 100°C, so there is a problem in that none of the RNases can be inactivated by heating.

[0036] FIG. 2 is a schematic diagram of a molecular diagnostic method according to one embodiment of the present invention, illustrating the reaction of components inside a tube. Referring to FIGS. 2(a) and 2(b), a sample containing cells or viruses is collected from the human body. The collected sample is added to the cell lysis and nucleic acid extraction composition to prepare a mixture. The RNase A inhibitor contained in the mixture inactivates RNase A contained in the sample. The mixture is then heated to inactivate all RNases other than RNase A, resulting in thermal inactivation. At the same time, cells are thermally lysed to extract nucleic acids, i.e., RNA or DNA, present inside the cells. Subsequently, when the extracted nucleic acids are mixed with primers, probes, and a premix to perform RT-PCR and PCR, the nucleic acid amplification time can be reduced.

[0037] According to one embodiment of the present invention, the RNase inhibitor may be derived from a protein. Specifically, the RNase inhibitor may be a large molecule. That is, the RNase inhibitor may be a large molecule derived from a protein, or may bind to an RNase to form a large molecule and prevent inhibition of the PCR reaction. More specifically, the RNase inhibitor may be one selected from mouse lung-derived RNase inhibitors, human placenta-derived RNase inhibitors, or a combination thereof. The mouse lung-derived RNase inhibitor may be nanohelix RI (RNase Inhibitor). The RNase inhibitors include Nanohelix HelixAyme RNase Inhibitor (RNI2000), Thermo Scientific RiboLock Inhibitor (EO0381), Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor (10777019), Takara Recombinant RNase Inhibitor (2313A), Invitrogen SUPERase In RNase Inhibitor (AM2694), Applied Biosystems RNase Inhibitor (N8080119), Roche Protector RNase Inhibitor (RNAINH-RO / 3335399001), Sigma-Aldrich Ribonuclease inhibitor human (R2520), Promega RNasin / RNasin Plus Ribonuclease Inhibitor, NEW ENGLAND BioLabs Inc. RNase Inhibitor, and Murine (M0314), NEW ENGLAND BioLabs Inc. RNase Inhibitor, Human Placenta (M0307), ABclonal Technology RNase Inhibitor, Mammalian (RK21401), BioVision RNaseOFFribonuclease Inhibitor (M1238), PCR Biosystems RiboShield RNase Inhibitor (PB30.23-02), Blirt RIBOPROTECT Hu RNase Inhibitor (RT35), highQu GmbH SecurRIN Advanced RNase Inhibitor (RNI0305), Enzynomics RNase Inhibitor (M007), Meridian Bioscience RiboSafe RNase Inhibitor (BIO-65027), QIAGEN RNase Inhibitor (Y9240L), Lucigen RiboGuard RNase Inhibitor (RG90925), Jena Bioscience RNase Inhibitor - recombinant (PCR392S), abm RNaseOFF Ribonuclease Inhibitor (G138), biotechrabbit RNase Inhibitor (BR0400901), BioFACT RNase Inhibitor (RI 152-20h), Canvax RNase Inhibitor (P0269), ShineGene RNasin (RNase Inhibitor) (ZP00801), TOYOBO RNase inhibitor (SIN-201), or a combination thereof. As mentioned above, RNase inhibitors derived from proteins are characterized by their large molecular size, and when such protein-derived RNase inhibitors with large molecular size are used, they can prevent the inhibition of PCR (polymerase chain reaction) in the molecular diagnostic process described below. However, RNase inhibitors derived from chemicals such as PVSA have small molecular size and may inhibit PCR (polymerase chain reaction) in the molecular diagnostic process. Guanidinium isothiocyanate (GuanidiniumChemically derived substances such as isothiocyanate (GITC) are known to inhibit RNases but also inhibit PCR reactions. Reducing agents such as beta-mercaptoethanol can also be used as RNase inhibitors, but reducing agents have drawbacks in terms of long-term storage and safety. Therefore, as mentioned above, by selecting a protein-derived RNase inhibitor, it is possible to prevent inhibition of PCR (polymerase chain reaction) in molecular diagnostic processes and reduce the time required for molecular diagnostic processes.

[0038] According to one embodiment of the present invention, the composition for cell lysis and nucleic acid extraction includes a buffer solution. As described above, the inclusion of a buffer solution in the composition for cell lysis and nucleic acid extraction can improve the reactivity of PCR (polymerase chain reaction) in molecular diagnostic processes and reduce the time required for molecular diagnostic processes.

[0039] According to one embodiment of the present invention, the buffer solution may have a pH of 6.0 to 9.0. Specifically, the buffer solution may have a pH of 6.1 to 8.9, pH of 6.2 to 8.7, pH of 6.3 to 8.6, pH of 6.4 to 8.5, pH of 6.5 to 8.4, pH of 6.6 to 8.3, pH of 6.7 to 8.2, pH of 6.8 to 8.1, pH of 6.9 to 8.0, pH of 7.0 to 7.9, pH of 7.1 to 7.8, pH of 7.2 to 7.7, pH of 7.3 to 7.6, or pH of 7.4 to 7.5. Adjusting the pH of the buffer solution within the above ranges can improve the efficiency of the cell lysis process and promote the reaction between the RNase inhibitor and RNase.

[0040] According to one embodiment of the present invention, the buffer solution may contain any one selected from glycerol, hydroxyethyl piperazine ethane sulfonic acid (HEPES), dithiothreitol (DTT), potassium chloride, and combinations thereof. As described above, adjusting the components contained in the buffer solution can improve the efficiency of the cell lysis process, promote the reaction between the RNase inhibitor and RNase, improve the reactivity of PCR (polymerase chain reaction) in molecular diagnostics, and reduce the time required for molecular diagnostics.

[0041] One embodiment of the present invention provides a method for cell lysis and nucleic acid extraction, comprising the steps of: adding a sample containing nucleic acids to the composition for cell lysis and nucleic acid extraction to prepare a mixture; performing a primary heating step to maintain the mixture at a temperature of 25°C or higher and 45°C or lower; and performing a secondary heating step to maintain the primarily heated mixture at a temperature of 75°C or higher and lower than 100°C.

[0042] The cell lysis and nucleic acid extraction method according to one embodiment of the present invention can improve the accuracy of molecular diagnostics by inactivating factors that inhibit the accuracy of polymerization chain reaction through heating during the nucleic acid extraction process.

[0043] 3 is a flowchart of a molecular diagnostic method according to one embodiment of the present invention. The method may include a sample collection step of collecting a biological sample from a human, a step of adding the collected sample to the composition for cell lysis and nucleic acid extraction to prepare a mixture (S110), a step of first heating (incubating) the mixture (S130), and a step of second heating (thermal lysis) the incubated mixture (S150).

[0044] According to one embodiment of the present invention, the method includes a 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 the composition for cell lysis and nucleic acid extraction contains a nucleic acid-containing sample, i.e., a biological specimen collected from a human, and thus contains RNase, an RNase inhibitor contained in the composition can be used to inactivate the RNase and protect RNA and other materials eluted from the cells from the RNase before the secondary heating (thermal lysis) step described below. Furthermore, the use of a specific RNase inhibitor can minimize unnecessary components, improve PCR sensitivity, and minimize the time required for molecular diagnosis.

[0045] The contents of the cell lysis and nucleic acid extraction method described in this specification that overlap with the composition for cell lysis and nucleic acid extraction will be omitted.

[0046] According to one embodiment of the present invention, the method may further include a step of collecting a biological sample from a human before the mixture preparation step (S110). Specifically, the biological sample from a human may be collected, without limitation, from blood, body fluids, saliva, or other samples containing nucleic acids, i.e., cells containing DNA and / or RNA. As described above, by collecting a biological sample from a human before the mixture preparation step (S110), molecular diagnostic targets, i.e., SAR-CoV-2 genes that cause novel coronavirus disease (COVID-19), can be amplified to facilitate molecular diagnosis.

[0047] According to one embodiment of the present invention, the method includes a first heating step (S130) in which the mixture is maintained at a temperature of 25°C to 45°C. Specifically, the first heating step may be an incubation step in which the RNase and the RNase inhibitor contained in the mixture are sufficiently bound to inactivate the RNase. 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. Most preferably, the temperature of the first heating step is maintained at 37°C. By adjusting the temperature of the primary heating (incubating) step within the above range, the reaction between the RNase and the RNase inhibitor can be promoted, thereby inactivating the RNase, and by inactivating the RNase before the cells are thermally lysed, degradation of RNA released from the cells can be prevented.

[0048] According to one embodiment of the present invention, the method further includes a second heating step (S150) of maintaining the first-heated mixture at a temperature of 75°C or higher but lower than 100°C. Specifically, the second heating step may be a cell thermal lysis step, which involves lysing the cells in a nucleic acid-containing sample, i.e., a cell-containing sample from a human biological specimen, to expose the DNA and / or RNA contained within the cells. Furthermore, the cell thermal lysis may be accompanied by thermal inactivation, which inactivates RNase, and may also simultaneously inactivate intracellular components that inhibit PCR. Specifically, the second heating step (S150) may simultaneously perform cell thermal lysis, RNase thermal inactivation, and intracellular component inactivation. Specifically, the secondary heating step may maintain the primarily heated mixture at 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 secondary heating step may maintain the primarily heated mixture at 94.5°C to 95.5°C or 95°C. By adjusting the temperature of the secondary heating (thermal lysis) step within the above range, it is possible to omit a separate additive for inhibiting RNase and maintain low concentrations of substances other than nucleic acids, thereby maximizing the removal of inhibitory factors, and since no separate additive for inhibiting RNase is included, PCR inhibition due to the additive can be prevented. In addition, by deactivating RNases other than RNase A, intracellular substances can be deactivated and cells can be thermally lysed to expose intracellular nucleic acids (DNA and / or RNA), thereby reducing the time required for molecular diagnosis.

[0049] According to one embodiment of the present invention, after nucleic acid extraction using the nucleic acid-containing sample, no separate elution or purification steps are required. As described above, by not requiring separate elution or purification steps after nucleic acid extraction, the time required for molecular diagnosis can be reduced, and costs can be reduced because no consumables or dedicated equipment for nucleic acid extraction are required.

[0050] According to one embodiment of the present invention, the primary heating step and the secondary heating step may each be performed for 1 minute to 30 minutes. Specifically, the primary heating step and the secondary heating step may 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 primary heating step and the secondary heating step may each be performed for 4.5 minutes to 5.5 minutes, or 5 minutes. By adjusting the time for each of the primary heating step and the secondary heating step within the above range, it is possible to maximize the inactivation of RNase and improve the effect of thermal lysis of cells.

[0051] According to one embodiment of the present invention, the concentration of the RNase inhibitor in the mixture may be 7.5 to 60.0 units / reaction, based on a volume of the mixture of 30 μL. The concentration of the RNase inhibitor in the mixture may be varied by increasing or decreasing the total volume. Specifically, the concentration of the RNase inhibitor in the mixture may be 7.5 to 60.0 units / reaction, 8.0 to 59.0 units / reaction, 9.0 to 58.0 units / reaction, 10.0 to 57.0 units / reaction, 15.0 to 55.0 units / reaction, 20.0 to 50.0 units / reaction, 25.0 to 45.0 units / reaction, or 30.0 to 40.0 units / reaction. More specifically, the concentration of the RNase inhibitor in the mixture may be 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. By adjusting the concentration of the RNase inhibitor in the mixture within the above ranges, it is possible to maximize the inactivation of RNase before the thermal lysis of the cells, prevent the degradation of RNA exposed from the cells after the thermal lysis of the cells, and minimize factors that inhibit PCR, thereby reducing the time required for molecular diagnosis.

[0052] As used herein, the unit "Unit / reaction (U / rxn)" refers to the amount of RNase inhibitor required to inhibit 5 ng of RNase A activity by 50% per reaction.

[0053] One embodiment of the present invention provides a molecular diagnostic method comprising the steps of adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by the cell lysis and nucleic acid extraction method, and amplifying the extracted nucleic acids by polymerase chain reaction.

[0054] A molecular diagnostic method according to one embodiment of the present invention can reduce the cost of molecular diagnostics by minimizing the dedicated equipment and consumables used for extraction.

[0055] Referring to Figure 3, one embodiment of the present invention includes a step (S170) of adding a solution containing primers and probes and a premix to a mixture containing nucleic acids extracted by the cell lysis and nucleic acid extraction method. Herein, a composition containing the solution containing primers and probes and the premix is ​​referred to as a "PCR sample." Specifically, by adding a PCR sample containing the solution containing primers and probes and the premix to a mixture containing nucleic acids extracted by the cell lysis and nucleic acid extraction method, components used for nucleic acid amplification are prepared, allowing nucleic acids to be easily amplified.

[0056] According to one embodiment of the present invention, the PCR sample includes a primer, and the base sequence of the primer is not particularly limited, but the primer can be the 2019-COVID primer sequence (N1) published by the Centers for Disease Control and Prevention (CDC). The sequence may be one published at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf, and any sequence that can perform a PCR reaction can be used without limitation.

[0057] According to one embodiment of the present invention, the PCR sample includes a probe, and the base sequence of the probe is not particularly limited, but the probe can be the 2019-COVID probe sequence (N1) published by the Centers for Disease Control and Prevention (CDC). The sequence may be one published at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf, and any sequence that can be used for PCR reactions can be used without limitation.

[0058] According to one embodiment of the present invention, the PCR sample contains a premix. The premix is ​​not particularly limited, but it is preferable to use Nanohelix's RealHelix™ qRT-PCR Kit [v6] (UDG System). Any premix that can perform a PCR reaction can be used without limitation.

[0059] According to one embodiment of the present invention, the adding step may involve adding a solution containing primers and probes and a premix, i.e., a PCR sample, to a well (or tube) containing a mixture containing nucleic acids extracted by the cell lysis and nucleic acid extraction method. As described above, by collecting a sample from the mixture and placing it in a separate tube without adding the PCR sample, all of the extracted nucleic acids can be used for nucleic acid amplification, minimizing loss of target genes and maximizing PCR performance. Furthermore, since a separate solution transfer process is not required, PCR preparation time can be reduced. Furthermore, by maximizing the use of nucleic acids exposed by the thermal lysis of the cells, dilution of the concentration due to the added PCR sample can be prevented, thereby reducing the time required for molecular diagnostics and preventing PCR inhibition due to additives.

[0060] According to one embodiment of the present invention, the molecular diagnostic method includes a step of amplifying the extracted nucleic acid by polymerase chain reaction (S190). Specifically, the step of amplifying by polymerase chain reaction may include sequentially performing RT-PCR (S191) and PCR (S193). As described above, amplifying the extracted nucleic acid by polymerase chain reaction can secure nucleic acid for molecular diagnosis and minimize the time required for molecular diagnosis.

[0061] According to one embodiment of the present invention, the molecular diagnostic method may amplify nucleic acids by polymerase chain reaction (PCR) without additional purification in wells (or tubes) to which a solution containing the primers and probes and a premix have been added. As described above, by amplifying nucleic acids by polymerase chain reaction without additional purification in wells to which a PCR sample has been added, the time required for molecular diagnostics can be minimized.

[0062] One embodiment of the present invention provides the use of a composition comprising an RNase inhibitor and a buffer solution for cell lysis and nucleic acid extraction.

[0063] One embodiment of the present invention provides a kit for cell lysis and nucleic acid extraction or molecular diagnostics, comprising a composition comprising an RNase inhibitor and a buffer solution.

[0064] One embodiment of the present invention provides the use of a composition comprising an RNase inhibitor and a buffer solution for the manufacture of a kit for cell lysis and nucleic acid extraction or a molecular diagnostic kit.

[0065] In one embodiment of the present invention, a composition containing an RNase inhibitor and a buffer solution is used to omit the solution purification and elution processes, and the dissolved solution is used to perform a polymerase chain reaction (PCR) to amplify nucleic acids for molecular diagnosis, thereby minimizing the time required for the process. Therefore, the composition can be used for cell lysis and nucleic acid extraction, and for kits for cell lysis and nucleic acid extraction or molecular diagnosis, and for the manufacture of the same.

[0066] Regarding the use of cell lysis and nucleic acid extraction, the kit, and the use for producing the kit according to one embodiment of the present invention, the composition for cell lysis and nucleic acid extraction, the molecular diagnostic method, the RNase inhibitor, and the buffer solution are as described above.

[0067] [Example] Hereinafter, the present invention will be described in detail with reference to examples. However, the examples of the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the examples described below. The examples in this specification are provided to more completely explain the present invention to those skilled in the art.

[0068] <Compounds used in Examples 1 to 4 and PCR conditions> The samples collected in Examples 1 to 4 below were collected using clinical swabs and stored in virus transport media, and the additional RNA samples used were purified target RNA.

[0069] Furthermore, the RNase inhibitor used in Examples 1 to 4 below was an RNase inhibitor derived from mouse lung (Rnase Inhibitor from Nanohelix), and the buffer solution was a mixture of glycerol, hydroxyethyl piperazine ethane sulfonic acid (HEPES), dithiothreitol (DTT), and potassium chloride.

[0070] The probe used in the PCR samples added for PCR in Examples 1 to 4 below was the 2019-COVID probe sequence (N1) published by the CDC (US Centers for Disease Control and Prevention), which is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. The primer used was the 2019-COVID primer sequence (N1) published by the CDC (US Centers for Disease Control and Prevention), which is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. Furthermore, the premix used was Nanohelix's RealHelix™ qRT-PCR Kit [v6] (UDG System).

[0071] The conditions for RT-PCR and PCR in Examples 1 to 4 below were (1) 10 minutes at 50°C, (2) 5 minutes at 95°C, and then (3) 10 seconds at 95°C and 30 seconds at 58°C. Only (3) was repeated 40 times, and the Ct (Threshold Cycle), which is the minimum threshold number of times at which the nucleic acid can be amplified and the results confirmed, was measured during this process.

[0072] Example 1 (Confirmation of the effect of thermal dissolution) FIG. 4 is a schematic diagram of the molecular diagnostic method according to Example 1 and a graph showing the Ct values ​​of Examples 1-1 and 1-2.

[0073] Specifically, Figure 4(a) is a schematic diagram of a molecular diagnostic method according to Example 1. Referring to Figure 4(a), in Example 1-1, a sample containing nucleic acids was collected, and distilled water was added to the collected sample, followed by thermal lysis at 95°C for 5 minutes. Then, a separately cultured RNA sample was added before RT-PCR, and RT-PCR and PCR were performed sequentially.

[0074] Example 1-2 was carried out in the same manner as Example 1-1, except that thermal lysis was not performed.

[0075] Figure 4(b) is a graph showing the Ct values ​​of Examples 1-1 and 1-2. Referring to Figure 4(b), it was confirmed that the Ct value of Example 1-1 was 0.5 lower than that of Example 1-2, which was confirmed to be due to the inactivation of PCR inhibitors (RNases other than RNase A) present with the sample during thermal lysis.

[0076] Example 2 (Confirmation of the effect of different types of RNAse inhibitors) FIG. 5 is a schematic diagram of the molecular diagnostic method according to Example 2 and a graph showing the Ct values ​​of Examples 2-1 to 2-4.

[0077] Specifically, Figure 5(a) is a schematic diagram of a molecular diagnostic method according to Example 2. Referring to Figure 5(a), in Example 2-1, a sample containing nucleic acids was collected, and distilled water was added to the collected sample, followed by thermal lysis at 95°C for 5 minutes. Then, a separately cultured RNA sample was added before RT-PCR, and RT-PCR and PCR were performed sequentially.

[0078] Example 2-2 was carried out in the same manner as in Example 2-1, except that the RNA sample was added to distilled water together with the specimen.

[0079] Example 2-3 was carried out in the same manner as in Example 2-2, except that distilled water containing the RNase inhibitor was used instead of distilled water.

[0080] Example 2-4 was carried out in the same manner as in Example 2-2, except that distilled water containing PVSA (polyvinylsulfonic acid), an RNase inhibitor derived from a chemical substance, was used instead of distilled water.

[0081] FIG. 5(b) is a graph showing the Ct values ​​of Examples 2-1 to 2-4. Referring to FIG. 5(b), in Example 2-1, the RNase contained in the sample was largely inactivated by thermal lysis, with only the remaining RNase A partially active. This resulted in a low Ct value for the RNA sample added immediately before RT-PCR, resulting in amplification. In contrast, in Example 2-2, the RNase contained in the sample partially active before thermal lysis, resulting in an increased Ct value of approximately 4.8 to 8 compared to Example 2-1, even when the order was changed under the same conditions. Furthermore, in Example 2-3, an RNase inhibitor was added along with the sample, which inactivated the RNase A, resulting in a lower Ct value of approximately 2.6 compared to Example 2-2. However, in Example 2-4, a chemical-derived RNase inhibitor was used instead of a protein-derived RNase inhibitor, resulting in a PCR-inhibiting effect, resulting in a Ct value 0.02 higher than Example 2-2.

[0082] Example 3 (Confirmation of the effect of buffer solution) FIG. 6 is a schematic diagram of the molecular diagnostic method according to Example 3 and a graph showing the Ct values ​​of Examples 3-1 and 3-2.

[0083] Figure 6(a) is a schematic diagram of a molecular diagnostic method according to Example 3. Referring to Figure 6(a), in Example 3-1, a sample containing nucleic acids was collected, and distilled water and a separately cultured RNA sample were added to the collected sample, followed by thermal lysis at 95°C for 5 minutes. RT-PCR and PCR were then performed sequentially.

[0084] Example 3-2 was carried out in the same manner as in Example 3-1, except that a buffer solution was used instead of distilled water.

[0085] Figure 6(b) is a graph showing the Ct values ​​of Examples 3-1 and 3-2. Referring to Figure 6(b), the Ct value of Example 3-2 was 1.8 lower than that of Example 3-1, confirming that changing only the buffer solution improved the PCR amplification effect.

[0086] Example 4 (Confirmation of the effects of thermal lysis, buffer solution, and type of RNase inhibitor) FIG. 7 is a schematic diagram of the molecular diagnostic method according to Example 4 and a graph showing the Ct values ​​of Examples 4-1 and 4-2.

[0087] Specifically, Figure 7(a) is a schematic diagram of a molecular diagnostic method according to Example 4. Referring to Figure 7(a), in Example 4-1, a sample containing nucleic acids was collected, and the collected sample was added to an RNA sample that had been separately incubated with distilled water, followed by thermal lysis at 95°C for 5 minutes. RT-PCR and PCR were then performed sequentially.

[0088] In Example 4-2, a sample containing nucleic acids was collected, added to distilled water, and subjected to thermal lysis at 95°C for 5 minutes. Then, a separately cultured RNA sample was added before RT-PCR, and RT-PCR and PCR were performed sequentially.

[0089] 7(b) is a graph showing the Ct values ​​of Examples 4-1 and 4-2. Referring to FIG. 7(b), it can be seen that in Example 4-1, the RNA sample is degraded by RNase present in the sample, resulting in a low concentration of RNA for nucleic acid amplification, and therefore a high Ct value is achieved. In contrast, in Example 4-2, the RNA sample is added immediately before RT-PCR for nucleic acid amplification, resulting in a very small amount of RNA inactivation, resulting in a high concentration of RNA and a low Ct value of 4.7.

[0090] Figure 8 is a schematic diagram of the molecular diagnostic methods according to Examples 4-3 to 4-6. Referring to Figure 8, Example 4-3 was carried out in the same manner as Example 4-1, except that a thermal dissolution process was added.

[0091] Example 4-4 was carried out in the same manner as in Example 4-3, except that an RNase inhibitor was added to the distilled water of Example 4-3.

[0092] Example 4-5 was carried out in the same manner as in Example 4-3, except that a buffer solution was added to the distilled water of Example 4-3.

[0093] Example 4-6 was carried out in the same manner as in Example 4-3, except that an RNase inhibitor and a buffer solution were added to the distilled water of Example 4-3.

[0094] It was confirmed that Example 4-3 exhibited a Ct value 0.5 lower than that of Example 4-1, which was confirmed to be due to the thermal inactivation of the RNase contained in the sample during the thermal lysis process.

[0095] Furthermore, it was confirmed that Example 4-4 exhibited a Ct value 3.1 lower than that of Example 4-1, which was confirmed to be due to the thermal inactivation effect confirmed in Example 4-3 and the RNase inhibitor's removal of RNase A and minimizing the amount of RNA that was degraded.

[0096] In addition, it was confirmed that Example 4-5 exhibited a Ct value 1.8 lower than that of Example 4-1, which confirmed that the buffer solution improved the PCR amplification effect.

[0097] In addition, it was confirmed that Example 4-6 achieved a Ct value 4.9 lower than that of Example 4-1. This was due to the thermal inactivation effect confirmed in Example 4-3, the effect of the RNase inhibitor inactivating RNase A confirmed in Example 4-4, and the effect of preventing PCR-inhibiting factors in the buffer solution confirmed in Example 4-5, thereby enabling the realization of a Ct value at the same level as Example 4-2.

[0098] <Production example> The specimens collected in the following manufacturing examples were collected using clinical swabs and then stored in virus transport media.

[0099] Furthermore, the RNase inhibitor used in the following preparation examples was an RNase inhibitor derived from mouse lung (nanohelix RNase Inhibitor), and the buffer solution was a mixture of glycerol, hydroxyethyl piperazine ethane sulfonic acid (HEPES), dithiothreitol (DTT), and potassium chloride. Furthermore, a mixture was prepared by mixing the collected specimen, RNase inhibitor, and buffer solution.

[0100] In the following manufacturing examples, the probe used in the PCR sample for PCR was the 2019-COVID probe sequence (N1) published by the CDC (US Centers for Disease Control and Prevention), which is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. The primer used was the 2019-COVID primer sequence (N1) published by the CDC (US Centers for Disease Control and Prevention), which is available at http: / / www.cdc.gov / coronavirus / 2019-ncov / downloads / rt-pcr-pane;-primer-probes.pdf. Furthermore, the premix used was Nanohelix's RealHelix™ qRT-PCR Kit [v6] (UDG System).

[0101] In the following production example, the conditions for RT-PCR and PCR were (1) 10 minutes at 50°C, (2) 5 minutes at 95°C, and then (3) 10 seconds at 95°C and 30 seconds at 58°C. Only (3) was repeated 40 times, and during this process, the Ct (Threshold Cycle), which is the minimum threshold number of times at which the nucleic acid can be amplified and the results confirmed, was measured.

[0102] Experimental example 1 (compared to PCR that includes a conventional extraction process) FIG. 9 is a graph showing the Ct values ​​of PCR including the RNA extraction and purification steps according to the prior art and the production example.

[0103] In a preparation example, a sample containing nucleic acids was collected, and distilled water, an RNase inhibitor, and a buffer solution were added to the collected sample, followed by thermal lysis at 95°C for 5 minutes. RT-PCR and PCR were then performed sequentially.

[0104] Referring to Figure 9, the results show a comparison of PCR performed using the conventional technology with the manufacturing example shown in Figure 1. It was confirmed that when a buffer solution containing an RNase inhibitor was added to the sample and the sample was subjected to thermal lysis, a Ct value at the same level as that of the conventional PCR method was obtained.

[0105] Experimental Example 2 (Confirmation of the effect of the concentration of RNAse inhibitor in the mixture in the first heating step) FIG. 10 is a graph showing the Ct value depending on the concentration of RNase inhibitor in the first heating step (incubating) of a preparation example.

[0106] Specifically, before performing RT-PCR and PCR in the Preparation Example, the mixture was subjected to a primary heating step (incubation) at 37°C for 5 minutes using various concentrations of the mixture, and Ct values ​​were measured for each concentration. More specifically, in the Preparation Example, a sample containing nucleic acid was collected, and the collected sample was added to distilled water, an RNase inhibitor, and a buffer solution, followed by thermal lysis at 95°C for 5 minutes. Then, the mixture was subjected to a primary heating step (incubation) at 37°C for 5 minutes using various concentrations of the mixture, followed by RT-PCR and PCR. The concentrations varied: 0 Unit / Reaction (U / rxn), 7.5 U / rxn, 15 U / rxn, 22.5 U / rxn, 30 U / rxn, 37.5 U / rxn, 45 U / rxn, and 52.5 U / rxn, and Ct values ​​were measured for each concentration.

[0107] Referring to Figure 10, it was confirmed that a high Ct value was achieved at 0U / rxn because no RNase inhibitor was included. From 7.5U / rxn to 45U / rxn, it was confirmed that the Ct value decreased with increasing RNase inhibitor concentration. However, in the case of 52.5U / rxn, it was confirmed that the Ct value increased with increasing RNase inhibitor concentration, but was lower than the Ct value at 7.5U / rxn.

[0108] Experimental example 3 (Confirming the effect of the temperature of the first heating step) FIG. 11 is a graph showing the Ct value as a function of the temperature in the first heating step in the production examples.

[0109] Specifically, before performing RT-PCR and PCR in the Preparation Example, the concentration of RNase inhibitor in the Preparation Example mixture was fixed at 30 U / rxn, and the mixture was subjected to a 5-minute primary heating step (incubation) while varying the temperature, and Ct values ​​were measured at each temperature. More specifically, in the Preparation Example, a sample containing nucleic acids was collected, and the collected sample was added to distilled water, RNase inhibitor, and a buffer solution, followed by thermal lysis at 95°C for 5 minutes. Then, the concentration of the Preparation Example mixture was fixed at 30 U / rxn, and the mixture was subjected to a 5-minute primary heating step (incubation) while varying the temperature, followed by RT-PCR and PCR. The temperatures were 25°C, 37°C, 45°C, and 60°C, and Ct values ​​were measured for each temperature.

[0110] Referring to Figure 11, it was confirmed that the Ct value remained constant at temperatures between 25°C and 37°C. Thereafter, the Ct value increased at temperatures above 45°C, confirming that the RNase inhibitor was inhibited when the temperature was 45°C or higher.

[0111] Therefore, the composition for cell lysis and nucleic acid extraction, the nucleic acid extraction method using the same, and the molecular diagnostic method using the same according to one embodiment of the present invention include an RNase inhibitor in the composition for nucleic acid extraction and inactivate RNase by heating, thereby eliminating a separate nucleic acid purification process, shortening the overall experiment time, and minimizing RNA damage to improve PCR performance.

[0112] As described above, the present invention has been described using limited examples, but the present invention is not limited thereto, and it goes without saying that various modifications and variations can be made by a person having ordinary skill in the art to which the present invention pertains within the technical spirit of the present invention and the equivalent scope of the claims set forth below. [Industrial Applicability]

[0113] The composition for cell lysis and nucleic acid extraction of the present invention can minimize the time required for the process of amplifying nucleic acids and performing molecular diagnosis by performing a polymerase chain reaction (PCR) using the lysed cell solution, omitting the separate purification and elution processes of the cell lysate solution. Furthermore, the composition can improve the accuracy of molecular diagnosis by inactivating factors that inhibit the accuracy of the PCR through heating during the nucleic acid extraction process, and therefore has industrial applicability. [Explanation of symbols]

[0114] S10: Sample collection step S30: Extraction step S31: Dissolution step S33: Elution step S50: Purification step S70:RT-PCR S90:PCR S110: Preparation of a mixture containing a specimen S130: First heating (incubating) step S150: Secondary heating (thermal melting) step S170: PCR sample addition step S190: Nucleic acid amplification step S191:RT-PCR S193:PCR

Claims

1. an RNase inhibitor and a buffer solution, the concentration of the RNase inhibitor in the composition is 1.0 Unit / μL or more and 1.75 Unit / μL or less; The buffer solution contains any one selected from glycerol, hydroxyethylpiperazineethanesulfonic acid (HEPES), dithiothreitol (DTT), potassium chloride, and combinations thereof; The RNase inhibitor is an inhibitor that inhibits RNase A derived from a protein. Compositions for cell lysis and nucleic acid extraction.

2. The buffer solution has a pH of 6.0 or more and a pH of 9.0 or less. The composition for cell lysis and nucleic acid extraction according to claim 1.

3. Adding a sample containing nucleic acids to the composition for cell lysis and nucleic acid extraction of claim 1 to produce a mixture; a first heating step of maintaining the mixture at a temperature of 25°C or higher and 45°C or lower; and a second heating step of maintaining the first heated mixture at a temperature of 75°C or higher but lower than 100°C. Cell lysis and nucleic acid extraction methods.

4. The primary heating step and the secondary heating step are each performed for 1 minute or more and 30 minutes or less. The method for cell lysis and nucleic acid extraction according to claim 3.

5. 4. The method for cell lysis and nucleic acid extraction according to claim 3, wherein the concentration of the RNase inhibitor in the mixture is 7.5 Units / reaction or more and 60.0 Units / reaction or less, based on a volume of the mixture of 30 μL.

6. adding a solution containing primers and probes and a premix; amplifying the extracted nucleic acid by polymerase chain reaction; The method for cell lysis and nucleic acid extraction according to claim 3.

7. A kit for cell lysis and nucleic acid extraction, comprising the composition for cell lysis and nucleic acid extraction according to claim 1.

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