Molecular diagnostic methods using cell lysis compositions for nucleic acid extraction

Abstract

To provide a molecular diagnosis method using a composition for cytolysis for nucleic acid extraction.SOLUTION: The present invention relates to a molecular diagnosis method using a composition for cytolysis for nucleic acid extraction in which: by containing only Tween20 as a solution for extracting nucleic acid and applying a polymerase chain reaction to a mixture to which only a PCR buffer solution has been added for the polymerase chain reaction, the polymerase chain reaction can be performed without another nucleic acid extraction process to minimize the time for molecular diagnosis, and the cost of molecular diagnosis can be reduced by minimizing dedicated devices and consumables used for extraction.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a molecular diagnostic method using a cell lysis composition for nucleic acid extraction. Specifically, the present invention relates to a molecular diagnostic method using a cell lysis composition for nucleic acid extraction, which performs a polymerase chain reaction (PCR) on a mixture containing only Tween 20 as a solution for nucleic acid extraction and adding only a PCR buffer solution for performing the PCR, thereby performing the PCR without a separate nucleic acid extraction process, thereby minimizing the time required for molecular diagnosis and reducing the cost of molecular diagnosis by minimizing the dedicated equipment and consumables used for extraction. [Background technology]

[0002] Recently, there has been a growing demand for the manipulation and biochemical analysis of biological samples in order to treat or prevent human diseases by analyzing the causes of diseases at the genetic level based on the results of human genetic 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 the presence or absence of viral or bacterial infections, and forensic medicine.

[0003] On the other hand, for molecular diagnosis, it is common to extract nucleic acids, which are DNA or RNA containing genetic information, from the saliva or blood of a person infected with a virus or bacteria, and then amplify them to confirm whether or not the person is infected with the disease.

[0004] Figure 1 is a schematic diagram of a conventional polymerase chain reaction (PCR). Conventionally, to extract nucleic acids containing genetic information from a sample, the sample was first dispensed and then the nucleic acids contained within the sample were extracted. The nucleic acid extraction process took approximately 60 minutes, after which the nucleic acids were collected, mixed with PCR reagents, dispensed, and the nucleic acids were dispensed. PCR was then performed. The nucleic acids were then amplified and the results analyzed. Nucleic acid extraction required lysis, purification, and elution processes. However, the lysis, purification, and elution processes required specialized extraction equipment and consumables (e.g., plastic tools, magnetic beads, or solutions) for the extraction.

[0005] When nucleic acids are extracted using the above-mentioned conventional techniques, highly pure nucleic acids can be extracted. However, the extraction process takes a long time, which makes it unsuitable for diagnosis or examination in an emergency situation or in an emergency room. Furthermore, in a situation where national-level disease prevention is required due to the rapid spread of a virus, dedicated equipment and consumables for application and extraction must be continuously used, which results in high costs for diagnosis.

[0006] Therefore, in order to solve the above-mentioned problems, there is an urgent need to develop a technology that can simultaneously perform nucleic acid extraction and amplification by polymerase chain reaction (PCR) without a separate nucleic acid extraction process by using a specific composition. Summary of the Invention [Problem to be solved by the invention]

[0007] The technical problem to be solved by the present invention is to provide a molecular diagnostic method using a cell lysis composition for nucleic acid extraction, which uses a specific solution in the process of lysing cells to extract intracellular nucleic acids, and performs a polymerase chain reaction by mixing the components necessary for the polymerase chain reaction all at once, thereby eliminating the need for a separate nucleic acid extraction process and enabling nucleic acid extraction and amplification through polymerase chain reaction.

[0008] However, the problems to be solved by the present invention are not limited to those described above, and other problems not described will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0009] One embodiment of the present invention provides a molecular diagnostic method comprising the steps of preparing a mixture containing a cell lysis composition for nucleic acid extraction, the composition comprising Tween 20 and distilled water; a sample containing nucleic acid; a premix; and a solution containing primers and probes; and performing a polymerase chain reaction on the mixture to extract and amplify nucleic acid. [Effects of the Invention]

[0010] A molecular diagnostic method using a cell lysis composition for nucleic acid extraction according to one embodiment of the present invention can omit a separate nucleic acid extraction process and perform a polymerase chain reaction using the lysed solution, thereby minimizing the time required for the process of amplifying nucleic acids for molecular diagnosis and reducing the cost of molecular diagnosis by minimizing the amount of dedicated equipment and consumables used for nucleic acid extraction.

[0011] The effects of the present invention are not limited to those described above, and other effects not described will be clearly understood by those skilled in the art from the present specification and the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing a polymerase chain reaction according to the prior art. [Figure 2]1 is a flowchart of a molecular diagnostic method using a cell lysis composition for nucleic acid extraction according to one embodiment of the present invention. [Figure 3] 1 is a graph showing fluorescence values ​​according to cycles in the polymerase chain reaction of Experimental Examples 1 to 6. DETAILED DESCRIPTION OF THE INVENTION

[0013] Throughout this specification, when a part is described as "comprising" a certain element, this means that it can further include other elements, but not excluding other elements, unless otherwise specified.

[0014] The present invention will be described in further detail below.

[0015] One embodiment of the present invention provides a molecular diagnostic method comprising the steps of preparing a mixture containing a cell lysis composition for nucleic acid extraction, the composition comprising Tween 20 and distilled water; a sample containing nucleic acid; a premix; and a solution containing primers and probes; and performing a polymerase chain reaction on the mixture to extract and amplify nucleic acid.

[0016] A molecular diagnostic method using a cell lysis composition for nucleic acid extraction according to one embodiment of the present invention can omit a separate nucleic acid extraction process and perform a polymerase chain reaction using the lysed solution, thereby minimizing the time required for the process of amplifying nucleic acids for molecular diagnosis and reducing the cost of molecular diagnosis by minimizing the amount of dedicated equipment and consumables used for nucleic acid extraction.

[0017] Referring to Figure 1, conventional molecular diagnostic methods involve lysis, purification, and elution processes to extract nucleic acids such as DNA or RNA from cells, and then adding a PCR buffer to the eluted solution to perform PCR (polymerase chain reaction). The nucleic acids amplified by PCR are then typically used for diagnosis. However, these methods have drawbacks, such as requiring specialized equipment for the lysis, purification, and elution processes, and requiring the continuous use of various consumables, such as solutions and plastic wires, used in these processes.

[0018] Figure 2 is a flowchart of a molecular diagnostic method using a cell lysis composition for nucleic acid extraction according to one embodiment of the present invention. Referring to Figure 2, the present invention provides a simple method for simultaneously extracting and amplifying nucleic acids by performing step S11 of preparing a mixture containing a cell lysis composition, a sample containing nucleic acids, a premix, and a solution containing primers and probes, followed by step S13 of extracting and amplifying nucleic acids by performing a polymerase chain reaction on the mixture. Specifically, the present invention limits the type of solution used in the cell wall lysis process to a specific type, and uses a cell lysis composition containing only Tween 20 without adding commonly used solutions. The PCR buffer solution for PCR, i.e., a premix, primers, and probes, is added, and the nucleic acids can be extracted and amplified for molecular diagnosis by simply performing PCR without a separate nucleic acid extraction process.

[0019] According to one embodiment of the present invention, the cell lysis composition for nucleic acid extraction may be a mixture containing only Tween 20 and distilled water. More specifically, the cell lysis composition for nucleic acid extraction may be composed of Tween 20 (polysorbate 20) and distilled water. By adjusting the components of the cell lysis composition for nucleic acid extraction from the above, molecular diagnosis can be simplified, and the nucleic acid extraction process can be omitted, and nucleic acid extraction and amplification can be achieved simultaneously by PCR, thereby shortening the time required for molecular diagnosis.

[0020] According to one embodiment of the present invention, a method includes preparing a mixture containing a cell lysis composition for nucleic acid extraction containing Tween 20 and distilled water; a sample containing nucleic acid; a premix; and a solution containing primers and probes. Specifically, the cell lysis composition for nucleic acid extraction may contain only Tween 20 and distilled water, or the premix and the solution containing primers and probes may be a PCR buffer solution. By including the step of preparing the mixture as described above, PCR can be performed to simultaneously extract and amplify nucleic acids without a separate nucleic acid extraction process, thereby shortening the time required for molecular diagnosis.

[0021] According to one embodiment of the present invention, the method further comprises the step of extracting and amplifying nucleic acids by performing a polymerase chain reaction on the mixture. By performing a polymerase chain reaction on the mixture to extract and amplify nucleic acids as described above, nucleic acids for molecular diagnosis can be secured and the time required for molecular diagnosis can be minimized.

[0022] According to one embodiment of the present invention, a separate step of extracting nucleic acids from a sample containing the nucleic acids is not included. By not including a separate nucleic acid extraction step as described above, the time required for molecular diagnosis can be shortened and costs can be reduced by not using consumables or dedicated equipment for nucleic acid extraction.

[0023] According to one embodiment of the present invention, the concentration of Tween 20 may be 2.0 vol / vol% or more and less than 16.0 vol / vol%. Specifically, the concentration of Tween 20 may be 2.2 vol / vol% or more and 15.8 vol / vol% or less, 2.4 vol / vol% or more and 15.6 vol / vol% or less, 2.6 vol / vol% or more and 15.4 vol / vol% or less, 2.8 vol / vol% or more and 15.2 vol / vol% or less, 3.0 vol / vol% or more and 15.0 vol / vol% or less, 3.2 vol / vol% or more and 14.8 vol / vol% or less, 3.4 vol / vol% or more and 14.6 vol / vol% or less, 3.6 vol / vol% or more and 15.8 vol / vol% or less. The Tween 20 concentration may be from 1.0 to 14.4 vol / vol%, from 3.8 to 14.2 vol / vol%, from 4.0 to 14.0 vol / vol%, from 4.2 to 13.8 vol / vol%, from 6.0 to 12.0 vol / vol%, from 7.0 to 11.0 vol / vol%, from 8.0 to 10.0 vol / vol%, or from 8.0 to 9.0 vol / vol%. Adjusting the Tween 20 concentration within the above ranges can improve the cell lysis effect, thereby enabling nucleic acid extraction and amplification by PCR without additional processes, thereby reducing the time required for molecular diagnosis.

[0024] According to one embodiment of the present invention, the concentration of a mixture containing a sample for nucleic acid analysis, i.e., a sample containing nucleic acids, can be changed by adding the composition for cell lysis for nucleic acid extraction to the composition for cell lysis for nucleic acid extraction. Specifically, by mixing the same volume of the composition for cell lysis for nucleic acid extraction and the sample, the surfactant concentration in the mixture containing the sample can be reduced to half of the concentration in the composition for cell lysis for nucleic acid extraction. Throughout this specification, the term "sample containing nucleic acids" can refer to a sample containing nucleic acids, such as a sample containing DNA or RNA nucleic acids in cells or a sample containing nucleic acids in a state dissolved in cells.

[0025] According to one embodiment of the present invention, the volume ratio of the sample to the cell lysis composition may be 1:0.5 to 1:2.0. By adjusting the volume ratio of the sample to the cell lysis composition within the above range, the concentration of Tween 20 can be adjusted, thereby enabling rapid extraction of intracellular nucleic acids.

[0026] According to one embodiment of the present invention, the volume ratio of the premix to the solution containing the primers and probes may be 1:1 to 1:10. By adjusting the volumes of the premix and the solution containing the primers and probes within the above range, the effects of nucleic acid extraction and amplification can be maximized, thereby shortening the time required for molecular diagnosis.

[0027] According to one embodiment of the present invention, the volume ratio of the mixture from which nucleic acids have been extracted to the premix may be 1:0.5 to 1:2.0. By adjusting the volumes of the mixture from which nucleic acids have been extracted and the premix within the above range, the effects of nucleic acid extraction and amplification can be maximized, thereby shortening the time required for molecular diagnosis.

[0028] According to one embodiment of the present invention, the nucleic acid extraction and amplification step may be performed by precipitating the mixture at a temperature above 60°C and below 100°C. Specifically, the nucleic acid extraction and amplification step may be performed at a temperature of 61°C to 99°C, 62°C to 98°C, 63°C to 97°C, 64°C to 96°C, 65°C to 95°C, 66°C to 94°C, 67°C to 93°C, 68°C to 92°C, 69°C to 91°C, or 70°C to 90°C. More specifically, the nucleic acid extraction and amplification step is preferably performed at a temperature of 50°C to 100°C. Adjusting the temperature of the nucleic acid extraction and amplification step within the above range can improve the cell lysis effect, thereby reducing the time required for molecular diagnosis by amplifying nucleic acids by PCR without a separate nucleic acid extraction process.

[0029] According to one embodiment of the present invention, the method may further include a step of storing the mixture in an incubator after the step of preparing the mixture. By storing the mixture in an incubator after the step of preparing the mixture as described above, the nucleic acid extraction efficiency can be improved. As used herein, the term "incubator" refers to any device having an internal space and capable of controlling temperature, humidity, pressure, etc., and is not limited to the above name.

[0030] According to one embodiment of the present invention, the step of leaving the mixture in the incubator may be carried out for 1 minute to 10 minutes. By adjusting the time for which the mixture is left in the incubator within the above range, the effect of lysing the cells can be improved.

[0031] According to one embodiment of the present invention, after the step of extracting and amplifying nucleic acids by performing a polymerase chain reaction on the mixture, a step of analyzing the solution from which the nucleic acids have been extracted and amplified can be further included. By including the analysis step as described above, the time required for the process of amplifying nucleic acids for molecular diagnosis can be minimized, and the cost of molecular diagnosis can be reduced by minimizing the need for dedicated equipment and consumables used for nucleic acid extraction.

[0032] The present invention will be described in detail below with reference to examples. However, the examples of the present invention can be modified into various different 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.

[0033] Experimental Example 1 To amplify the target gene, 16S rRNA, from E. coli DH3, the test subject was extracted using a spin-column extraction method. Specifically, the test subject E. coli DH3 was collected, and then subjected to lysis, purification, and elution processes using the conventional Qiagen extraction method to extract the 16S rRNA.

[0034] An intercalating dye (Ssofast pol. EvaGreen, Green) was then added to the extracted mixture and pre-reacted at 95°C for 2 minutes. The target gene, 16S rRNA, was amplified by 50 cycles of maintaining the temperature at 95°C for 10 seconds and then at 61°C for 10 seconds. Fluorescence was measured at each cycle, and the threshold cycle (Ct), the minimum number of cycles at which the nucleic acid could be amplified and the results confirmed, was measured and summarized in Table 1 below.

[0035] Experimental Example 2 To amplify the target gene, 16S rRNA, from E. coli DH3, the gene was extracted using the E3 extraction method. More specifically, the E3 extraction method is an automated nucleic acid extraction method, which can be broadly divided into an extraction reagent (E3 Nucleic Acid Extraction Kit) and an extraction device. This method extracts nucleic acids (DNA / RNA) from human samples using iron oxide-coated magnetic beads. The reagent tube has four wells, each containing a lysis buffer, a washing buffer, and an elution buffer. The high concentration of chaotropic salt contained in the lysis buffer and washing buffer disrupts cell membranes, separating nucleic acids from proteins. In this environment, the magnetic beads adsorb the negatively charged nucleic acids. The nucleic acids adsorbed to the magnetic beads are transferred sequentially to each column of the tube by the magnetic rod of the nucleic acid extraction device (E3 device) and undergo several washing steps. Finally, the elution buffer separates the nucleic acids adsorbed to the magnetic beads, and the magnetic rod removes only the magnetic beads, leaving only the pure nucleic acids in the elution buffer. 16S rRNA was extracted as described above.

[0036] An intercalating dye (Ssofast pol. EvaGreen, Green) was then added to the extracted mixture and pre-reacted at 95°C for 2 minutes. The target gene, 16S rRNA, was amplified by maintaining the temperature at 95°C for 10 seconds and then maintaining the temperature at 61°C for 10 seconds, 50 times. Fluorescence was measured at each cycle. The threshold cycle (Ct), the minimum number of cycles at which the nucleic acid could be amplified and the results confirmed, was measured during this process. The difference in Ct from Experimental Example 1, ΔCt, was calculated and summarized in Table 1 below.

[0037] Experimental Example 3 To amplify 16S rRNA, the target gene of E. coli DH3, a cell lysis composition for nucleic acid extraction containing Tween 20 and distilled water was prepared at a concentration of 10 vol / vol%, and a sample containing the test subject was added to the composition to prepare a mixture with the Tween 20 concentration adjusted to 5 vol / vol%. After incubation at room temperature for 5 minutes, the mixture was centrifuged at 15,000 g for 15 minutes.

[0038] An intercalating dye (Ssofast pol. EvaGreen, Green) was then added to the extracted mixture and pre-reacted at 95°C for 2 minutes. The target gene, 16S rRNA, was amplified by maintaining the temperature at 95°C for 10 seconds and then maintaining the temperature at 61°C for 10 seconds, 50 times. Fluorescence was measured at each cycle. The threshold cycle (Ct), the minimum number of cycles at which the nucleic acid could be amplified and the results confirmed, was measured during this process. The difference in Ct from Experimental Example 1, ΔCt, was calculated and summarized in Table 1 below.

[0039] Experimental Example 4 In Experimental Example 3, except that the concentration of Tween 20 in the cell lysis composition for nucleic acid extraction was adjusted to 20 vol / vol% and the concentration of Tween 20 in the mixture was adjusted to 10 vol / vol%, the fluorescence value and Ct (Threshold Cycle) were measured for each cycle in the same manner as in Experimental Example 3, and ΔCt, which is the difference in Ct from Experimental Example 1, was calculated and summarized in Table 1 below.

[0040] Experimental Example 5 In Experimental Example 3, except that Triton X-100 was used instead of Tween 20 as the cell lysis composition for nucleic acid extraction, the fluorescence value and Ct (Threshold Cycle) were measured for each cycle in the same manner as in Experimental Example 3, and ΔCt, which is the difference in Ct from Experimental Example 1, was calculated and summarized in Table 1 below.

[0041] Experimental Example 6 In Experimental Example 4, except that Triton X-100 was used instead of Tween 20 as the cell lysis composition for nucleic acid extraction, the fluorescence value and Ct (Threshold Cycle) were measured for each cycle in the same manner as in Experimental Example 4, and ΔCt, which is the difference in Ct from Experimental Example 1, was calculated and summarized in Table 1 below.

[0042] [Table 1]

[0043] Figure 3 is a graph showing fluorescence values ​​according to cycles in the CPRC of Experimental Examples 1 to 6. Referring to Figure 3 and Table 1, it was confirmed that Experimental Example 3, which used Tween 20 at a concentration of 10 vol / vol% as a cell lysis composition for nucleic acid extraction, was comparable to the conventional nucleic acid extraction methods of Experimental Examples 1 and 2. However, since Experimental Example 3 did not perform the purification and elution processes in the nucleic acid extraction process, it was confirmed that the time required for molecular diagnosis was shorter than that of Experimental Examples 1 and 2.

[0044] In contrast, in Experiments 5 and 6, in which Triton X-100 was used as a cell lysis composition for nucleic acid extraction, it was confirmed that the cell walls dissolved more slowly within the same time period, resulting in an increase in Ct value at the same fluorescence value (RFU).

[0045] Furthermore, it was confirmed that Experimental Example 4, which used Tween 20 at a concentration of 20 vol / vol%, had a higher Ct value for the same fluorescence value (RFU) compared to Experimental Example 3, which used Tween 20 at a concentration of 10 vol / vol%.

[0046] Through the above Experimental Examples 1 to 3, it was confirmed that Tween 20 is suitable as a cell lysis composition for extracting nucleic acids from cells.

[0047] The effects of a conventional molecular diagnostic method and a molecular diagnostic method according to an embodiment of the present invention are compared below.

[0048] Comparative Example 1 To amplify genes contained in the test subjects, M. tuberculosis, M. avium, and M. intracellulare, the genes were extracted using a spin-column extraction method. Specifically, the test subjects, M. tuberculosis, M. avium, and M. intracellulare, were collected, and then the genes were extracted using the conventional Qiagen extraction method, with lysis, purification, and elution processes.

[0049] Thereafter, 16 μl of the extracted mixture was mixed with 4 μl of a primer / probe mixture that can specifically detect M. tuberculosis and NTM (nontuberculous mycobacteria; including M. avium and M. intracellulare) and Taq polymerase premix (Realhelix). TM 20 μl of qPCR Kit (Nanohelix, Korea) was added to the mixture, and a pre-reaction was carried out at 95°C for 5 minutes. Thereafter, the mixture was maintained at 95°C for 10 seconds, followed by 60°C for 40 seconds, which was repeated 40 times to amplify the gene. During this process, the Ct (Threshold Cycle), which is the minimum threshold cycle at which the nucleic acid can be amplified and the results can be confirmed, was measured and summarized in Table 2 below.

[0050] Comparative Example 2

[0051] To amplify genes contained in the target M. tuberculosis, M. avium, and M. intracellulare, a cell lysis composition for nucleic acid extraction was prepared by measuring and mixing the volumes of Tween 20 and distilled water to a concentration of 5.0 vol / vol%. Then, 20 μl of a cell-containing sample was mixed with 20 μl of the cell lysis composition for nucleic acid extraction to prepare a sample-containing mixture (detergent) with a Tween 20 concentration of 2.5 vol / vol%. The cells were lysed at 90°C for 5 minutes to extract the nucleic acids contained within the cells.

[0052] Thereafter, 16 μl of the extracted mixture was mixed with 4 μl of a primer / probe mixture that can specifically detect M. tuberculosis and NTM (nontuberculous mycobacteria; including M. avium and M. intracellulare) and Taq polymerase premix (Realhelix). TM 20 μl of qPCR Kit (Nanohelix, Korea) was added to the mixture and pre-reacted at 95°C for 5 minutes. This was followed by 40 cycles of holding at 95°C for 10 seconds and then at 60°C for 40 seconds, amplifying the target gene IS6110 and the ITS region. Fluorescence was measured at each cycle, and the threshold cycle (Ct), the minimum number of cycles at which the nucleic acid could be amplified and the results confirmed, was measured. ΔCt, the difference in Ct from Experimental Example 1, was calculated and summarized in Table 2 below.

[0053] Example 1 To amplify genes contained in the target M. tuberculosis, M. avium, and M. intracellulare, a cell lysis composition for nucleic acid extraction was prepared by measuring and mixing the volumes of Tween 20 and distilled water to a concentration of 12.5 vol / vol%. Then, 20 μl of a cell-containing sample was mixed with 20 μl of the cell lysis composition for nucleic acid extraction to prepare a sample-containing mixture with a Tween 20 concentration of 6.25 vol / vol% (the concentration was adjusted so that the final Tween 20 concentration in the following PCR reaction solution, 40 μl in total, was 2.5 vol / vol%).

[0054] Thereafter, 16 μl of the mixture was added to Taq polymerase premix (Realhelix TM 20 μl of a qPCR kit (Nanohelix, Korea) and 4 μl of a solution containing primers and probes capable of specifically detecting M. tuberculosis and NTM (nontuberculous mycobacteria, including M. avium and M. intracellulare) were added and pre-reacted at 95°C for 5 minutes. Gene amplification was then performed by repeating 40 cycles of maintaining the temperature at 95°C for 10 seconds and then at 60°C for 40 seconds. The Ct (Threshold Cycle), the minimum threshold cycle at which the nucleic acid could be amplified and the results confirmed, was measured and summarized in Table 2 below.

[0055] [Table 2]

[0056] Furthermore, for each test subject identified from Table 1, ΔCt, which is the difference in Ct between Comparative Example 1, Comparative Example 2, and Example 1, was calculated and summarized in Table 3 below.

[0057] [Table 3]

[0058] Referring to Tables 2 and 3, it was confirmed that there was an average difference of 0.52 Ct between Comparative Example 1, in which nucleic acids were extracted using a conventional Qiagen spin-column extraction method and then amplified by PCR, and Example 1, which is an embodiment of the present invention. It was also confirmed that there was an average difference of 0.47 Ct between Comparative Example 2, in which nucleic acids were extracted using a cell lysis composition containing only Tween 20 and then amplified by PCR, and Example 1, which is an embodiment of the present invention.

[0059] In conclusion, Example 1 according to one embodiment of the present invention exhibits a Ct value similar to or superior to that of Comparative Examples 1 and 2, which are conventional techniques, but since there is no nucleic acid extraction process, it was confirmed that the time required for molecular diagnosis is reduced compared to conventional techniques, and that the cost of molecular diagnosis is reduced because no consumables are used.

[0060] Therefore, in one embodiment of the molecular diagnostic method using a cell lysis composition for nucleic acid extraction according to the present invention, the solution for nucleic acid extraction contains only Tween 20, and the components necessary for the polymerase chain reaction are mixed at once to perform the polymerase chain reaction, thereby omitting a separate nucleic acid extraction process and achieving nucleic acid extraction and amplification through the polymerase chain reaction.

[0061] Although the present invention has been described above using limited examples, it is to be understood that the present invention is not limited thereto, and that various modifications and variations can be made by those skilled in the art within the scope of the technical concept of the present invention and the scope of the claims set forth below. [Explanation of symbols]

[0062] S11: Mixture production step S13: Nucleic acid extraction and amplification step

Claims

1. A step of preparing a mixture including: a cell lysis composition for nucleic acid extraction, which comprises Tween 20 at a concentration of 10.0 to 12.5 vol / vol% and distilled water; a sample containing nucleic acids; a premix; and a solution containing primers and probes; and performing a polymerase chain reaction on the mixture to extract and amplify nucleic acids, wherein the cells are derived from bacteria (with the proviso that the bacteria are E. coli, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare).

2. 2. The method of claim 1, wherein the volume ratio of the sample to the cell lysis composition is 1:0.5 to 1:2.

0.

3. 2. The method of claim 1, wherein the volume ratio of the premix to the solution containing the primers and probes is 1:1 to 1:

10.

4. 2. The method of claim 1, wherein the volume ratio of the mixture containing the cell lysis composition and the sample containing nucleic acid to the premix is ​​1:0.5 to 1:2.

0.

5. 10. The method of claim 1, wherein the steps of extracting and amplifying nucleic acids are carried out at a temperature greater than 60°C and less than or equal to 100°C.

6. After the step of preparing the mixture, The method of claim 1 , further comprising the step of placing the mixture in an incubator.

7. The method of claim 6, wherein the step of leaving in the incubator is carried out for a period of at least 1 minute and not more than 10 minutes.

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