Method for extracting nucleic acids from biological samples in an automated liquid handling system

Abstract

The present disclosure relates to a method for extracting nucleic acids from biological samples in an automated liquid handling system, more specifically, a method for extracting nucleic acids from biological samples in large quantities and with improved sensitivity without purification using a cell lysis composition in an automated liquid handling system. The method of the present disclosure can obtain nucleic acids without complex purification steps. Accordingly, the method of the present disclosure reduces the cost and time for molecular diagnostics by eliminating the need for reagents and tools previously used to purify nucleic acids.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to Korean Patent Application No. 10-2024-0068067, filed with the Korean Intellectual Property Office on May 24, 2024, the entire disclosure of which is incorporated herein by reference.

[0003] Technology field

[0004] The present disclosure relates to a method for extracting nucleic acids from a biological sample in an automated liquid handling system, and more specifically, a method for extracting nucleic acids from a biological sample in large quantities without purification and with improved sensitivity using a cell lysis composition in an automated liquid handling system. Background Technology

[0005] Molecular diagnostics is a rapidly growing field within the in vitro diagnostics market for the early diagnosis of diseases. In particular, nucleic acid-based methods are being used to diagnose causative genetic factors associated with viral or bacterial infections due to their high specificity and sensitivity.

[0006] Most nucleic acid-based methods involve the amplification of a target nucleic acid (e.g., viral or bacterial nucleic acid). As a representative example, the polymerase chain reaction (PCR) involves repeated cycles of denaturation of double-stranded DNA, annealing of primers into a DNA template, and primer extension by DNA polymerase (Mullis et al., U.S. Patents No. 4,683,195, 4,683,202 and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354). Alternatively, various methods such as LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification), and RCA (Rolling-Circle Amplification) have been proposed.

[0007] The real-time PCR method includes the steps of collecting a sample from a subject, such as a nasopharyngeal or oropharyngeal swab sample, storing it in a transport medium, and then extracting and purifying nucleic acids prior to real-time PCR.

[0008] The extraction and purification of nucleic acids are critical for removing potential PCR inhibitors and ensuring reliable results. However, these processes are time-consuming and costly, and require skilled technicians for accurate execution, making them a bottleneck in the overall diagnostic testing workflow.

[0009] To address these problems, an automated high-throughput system (Cameron, A. et al., J. Clin. Microbiol. 59 , 2; Kim, TY et al. Ann. Lab. Med. 42 ​​​, 473-477; Ji, L. et al. Environ. Sci. Technol. 56 , 13398-13407), and magnetic beads (Pham, XH et al. Chem. Asian J. 12 , 1883-1888; Pang, Y. et al. Biosens. Bioelectron. 79 , 574-580), solid-phase extraction (Nacham, O. et al., Anal. Chem. 88 , 7813-7820; Varona, M. et al., Anal. Chem. 90 , 6922-6928), and microfluidic device (Obino, D. et al. Sens. (Basel) 21 , 3059; Jin, CE et al. Anal. Chem. 89 , 7502-7510; Wen, J. et al., Anal. Chem. 80 A method using , 6472-6479) was proposed.

[0010] In addition to these technological advancements, there have been efforts to optimize protocols or introduce extraction-free methods (Fomsgaard, AS & Rosenstierne, MW Euro Surveillance. 25 , 14; Villota, SD et al. , J. Virol. Methods 298 , 114302; Lownik, JC, Way, GW, Farrar, JS & Martin, RK; J. Mol. Diagn. 23 , 1671-1679; Kim, Y.K. & Chang, SH; J. Virol. Methods 296 , 114217; Visseaux, B. et al., J. Virol. Methods 291 ​​​​​​​​​​​​​​​​​​​, 11408; Smyrlaki, I. et al. , Nat. Commun. 11 , 4812).

[0011] No-extraction methods can simplify testing and expedite processing by avoiding the time-consuming and labor-intensive extraction step. Examples of these include diluting the sample 1:1 to reduce inhibitors that may interfere with PCR, heating the sample to about 100°C, treating the sample with a soluble substance, or using a combination thereof.

[0012] Despite these advantages, the extraction-free method suffers from low sensitivity due to the presence of PCR inhibitors and the degradation of nucleic acids in the sample.

[0013] A new extraction-free method to solve this problem is required in the industry.

[0014] Throughout this specification, numerous cited literature and patent literature are referenced and their citations are indicated. The disclosures of the cited literature and patents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. The problem to be solved

[0015] The inventors have made diligent research efforts to develop a novel nucleic acid extraction method capable of reducing the time and labor required for nucleic acid extraction while simultaneously improving low sensitivity.

[0016] As a result, the inventors confirmed that by preheating a cell lysis composition in an automated liquid handling system, mixing the cell lysis composition with a sample to obtain a mixture, and then heating the mixture, nucleic acids can be effectively extracted without purification, and the extracted nucleic acids can be used in a nucleic acid amplification reaction with high sensitivity.

[0017] ​Accordingly, the object of the present invention is to provide a method for extracting nucleic acids from a biological sample in an automated liquid handling system.

[0018] Other objects and advantages of the present disclosure will become more apparent from the following detailed description together with the appended claims. means of solving the problem

[0019] In one aspect of the present disclosure, a method for extracting nucleic acids from a biological sample in an automated liquid handling system is provided, comprising the following steps:

[0020] (i) a step of dispensing a cell lysis composition into an empty reaction vessel mounted on a heating element in an automated liquid handling system, wherein the cell lysis composition serves to lyse cells in a biological sample;

[0021] (ii) a step of pre-incubating the cell lysis composition at 50°C or higher using a heating element;

[0022] (iii) a step of preparing a mixture by dispensing a biological sample into the reaction vessel; and

[0023] (iv) a step of extracting nucleic acids from a biological sample by incubating the above mixture at 50°C or higher using a heating element,

[0024] Steps (i)-(iv) are controlled by a controller within an automated liquid handling system, and steps (i) and (iii) are performed by a pipetting channel.

[0025] In a specific embodiment, the biological sample is a swab, saliva, or a mixture thereof from a subject.

[0026] In a specific embodiment, the biological sample is Streptococcus piogenes ( Streptococcus pyogenes ), Streptococcus equity subspecies equity ( Streptococcus equi subsp. Equi) , Streptococcus equi subspecies zueepidemicus ( Streptococcus equi subsp. zooepidemicus ) , Streptococcus disgalactiae subspecies disgalactiae ( Streptococcus dysgalactiae subsp. dysgalactiae ) , Streptococcus disgalactiae subspecies equityimilis ( Streptococcus dysgalactiae subsp. equisimilis ) , Streptococcus cannis ( Streptococcus canis ) , Arcanobacterium hamolitiscum Arcanobacterium haemolyticum It contains or is suspected of containing ), and a combination thereof.

[0027] In a specific embodiment, the heating element serves to heat the reaction vessel mounted thereon to a predetermined temperature while shaking it.

[0028] In a specific embodiment, the reaction vessel is a deep-well plate or a well plate.

[0029] In a specific embodiment, the cell lysis composition comprises (a) guanidine hydrochloride (GuHCl); (b) cetyltrimethylammonium bromide (CTAB); and (c) polyethylene glycol (PEG) having a molecular weight of 200 to 1000 Da.

[0030] In a specific embodiment, the cell lysis composition comprises an amount of 30 to 300 mM of GuHCl, an amount of about 0.05 to about 0.5 weight% of CTAB based on the total weight of the cell lysis composition, and an amount of about 5 to about 80 weight% of PEG based on the total weight of the cell lysis composition.

[0031] In a specific embodiment, the cell lysis composition further comprises a detergent, a chelating agent, a buffer, or a combination thereof.

[0032] In a specific embodiment, the cell lysis composition of step (i) is dispensed in an amount of 10 to 100 μL.

[0033] In a specific embodiment, the method further comprises the step of heating an empty reaction vessel by a heating element in an automated liquid handling system prior to step (i).

[0034] In a specific embodiment, the heating element of step (ii) is controlled by a controller so that its temperature increases to 95°C or higher.

[0035] In a specific embodiment, the heating element of step (ii) is controlled by a controller so that its temperature is maintained at 95°C or higher.

[0036] In a specific embodiment, the pre-incubation of step (ii) is performed for at least 5 minutes.

[0037] In a specific embodiment, the biological sample of step (iii) is dispensed in an amount of 10 to 100 μL.

[0038] In a specific embodiment, the biological sample of step (iii) is dispensed in an amount equal to the cell lysis composition dispensed in step (i).

[0039] In a specific embodiment, the heating element of step (iv) is controlled by a controller so that its temperature increases to 95°C or higher.

[0040] In a specific embodiment, the extracted nucleic acid is applied directly to a nucleic acid amplification reaction without further purification.

[0041] In a specific embodiment, the nucleic acid amplification reaction is PCR, real-time PCR, or LAMP (Loop Mediated Isothermal Amplification). Effects of the invention

[0042] The features and advantages of the present invention are summarized as follows:

[0043] (a) The method of the present disclosure can obtain nucleic acids without complex purification steps. Therefore, the method of the present disclosure reduces the cost and time for molecular diagnostics by eliminating the need for reagents and tools used to purify nucleic acids.

[0044] (b) The method of the present disclosure can extract nucleic acids in large quantities using an automated liquid handling system. Accordingly, the method of the present disclosure can reduce human error and labor, and is useful in large hospitals, contract testing laboratories, and laboratories.

[0045] (c) When using reaction vessels with low thermal conductivity, such as deep-well plates, in automated liquid handling systems, it is difficult to raise the temperature of the cell lysis composition or the mixture of the cell lysis composition and the sample within the reaction vessel to near 100°C. Therefore, conventional nucleic acid extraction methods by heating cannot achieve high nucleic acid extraction efficiency. In contrast, the method of the present disclosure can achieve high nucleic acid extraction efficiency through pre-incubation of the cell lysis composition at 50°C or higher, even without raising the temperature of the cell lysis composition or the mixture of the cell lysis composition and the sample to near 100°C.

[0046] (d) The method of the present invention can further improve extraction efficiency by using a cell lysis composition having a specific composition. Specific details for implementing the invention

[0047] The inventors have made diligent research efforts to develop a novel nucleic acid extraction method capable of reducing the time and labor required for nucleic acid extraction while simultaneously improving low sensitivity.

[0048] As a result, the inventors confirmed that by preheating a cell lysis composition in an automated liquid handling system, mixing the cell lysis composition with a sample to obtain a mixture, and then heating the mixture, nucleic acids can be effectively extracted without purification, and the extracted nucleic acids can be used in a nucleic acid amplification reaction with high sensitivity.

[0049] I. justice

[0050] The following defines terms used in relation to the present invention.

[0051] As used herein, the term “biological sample” means an analyte obtained from a subject that contains or is suspected of containing the nucleic acid to be detected. Examples thereof include, but are not limited to, viruses, bacteria, tissues, cells, blood (including whole blood, plasma, and serum), lymph, bone marrow fluid, saliva, sputum, swabs, aspirations, milk, urine, feces, ocular fluid, semen, brain extracts, cerebrospinal fluid, joint fluid, thymic fluid, bronchial lavage fluid, ascites fluid, and amniotic fluid. In certain embodiments, the biological sample is a swab, saliva, or a mixture thereof from a subject. In certain embodiments, the smear may be, but is not limited to, a throat swab or a nasal swab, such as a nasopharyngeal swab. The sample may be used interchangeably with “specimen” herein.

[0052] As used herein, the term "subject" refers to an individual suspected of containing the target nucleic acid (e.g., a target nucleic acid from a specific pathogen) to be detected using the method of the present disclosure. Examples of said subject include, but are not limited to, mammals such as dogs, cats, rodents, primates, and humans, and in particular, humans.

[0053] The terms “nucleic acid,” “nucleic acid sequence,” or “nucleic acid molecule” as used herein refer to single- or double-stranded deoxyribonucleotides or ribonucleotide polymers, and said nucleotides may include derivatives of natural nucleotides, non-natural nucleotides, or modified nucleotides that can function in the same way as naturally occurring nucleotides.

[0054] As used herein, the terms “target nucleic acid,” “target nucleic acid sequence,” or “target sequence” refer to a nucleic acid sequence whose presence is to be detected. The nucleic acid extracted by the method of the present invention may contain the nucleic acid to be detected, such as the nucleic acid of a specific organism, particularly a pathogen, or a mutated nucleic acid, which is referred to herein as the target nucleic acid. The presence of said target nucleic acid may be determined by various methods known to those skilled in the art, such as nucleic acid amplification-based molecular diagnostics (e.g., PCR, real-time PCR, digital PCR, LAMP, etc.), after nucleic acid extraction according to the present disclosure. The presence of a specific target nucleic acid in a biological sample may indicate the presence or absence of a mutation of the organism from which said target nucleic acid originates, such as a pathogen.

[0055] The present disclosure provides a method for obtaining nucleic acids in large quantities and conveniently for use in determining the subsequent presence of target nucleic acids, such as in molecular diagnostics and sequencing as described above.

[0056] As used herein, the term "automated liquid handling system" refers to a system that automatically performs repetitive liquid handling tasks that were previously performed manually in a laboratory. Typically, an automated liquid handling system serves to dispense a predetermined amount of liquid (e.g., reagents, samples, etc.) into a desired container for tasks such as pipetting, sample preparation, and microplate cleaning. Automated liquid handling systems can reduce human error, as well as working time and costs, and offer the advantage of enabling high-throughput operations.

[0057] Automated liquid handling systems are commercially available, examples of which are, but not limited to, Hamilton’s Microlab Prep, Microlab NIMBUS, Microlab STAR, and Microlab VANTAGE; Eppendorf’s epMotion 5070 and 5075 Automated Pipetting Systems, epMotion 5075 TMX, epMotion Automated Pipetting System, epMotion® 5075 LH Automated Pipetting Systems, and EpMotion® 96 Semi-Automated Electronic Pipette; Flow-Robotics’ flowbot® ONE Liquid Handling Robot; INTEGRA Biosciences’ ASSIST PLUS Pipetting Robot, MIRO CANVAS NGS Prep System, and ASSIST Pipetting Platform; Opentrons’ Opentrons Flex NGS Workstation and OT-2 Pipetting Robot; Biotage’s Biotage® Extrahera™ Automated Sample Processing System and Biotage® Extrahera™ LV-200; BRAND LHS Liquid Handling Station Flow and BRAND® Liquid Handling Station Pipetting Robot from BrandTech® Scientific; Corning® Lambda™ EliteMax Semi-automated Benchtop Pipettor from Corning;Hudson SOLO™ Automated Pipettor, NGS Library Prep Workcell, PlateCrane EX Robotic Arm Microplate Handler, PlateCrane VX Microplate Handling, Protean™ Workcell, and SOLO™ Plus Pipettor and Dispenser from Hudson Robotics; Metrohm 815 Robotic Titration Soliprep from Metrohm; LH-40 Nexera Prep Liquid Handler from Shimadzu; Thermo Scientific™ TriPlus™ RSH Autosampler and Liquid Handling System from Thermo Fisher Scientific; and the Andrew+ Pipetting Robot from Waters Andrew Alliance;

[0058] The automated liquid handling system described above may typically include various components such as a controller for controlling operation, a robotic arm or pipetting channel for dispensing liquid, a heating element, a cooling element, a shaking element, etc., which may be changed or adjusted depending on the application. In addition to the above components, additional components may be integrated into the automated liquid handling system if necessary.

[0059] The automated liquid handling system used in the method of the present disclosure comprises a controller that controls the entire step, a pipetting channel that aspirates and dispenses a cell lysis composition and a biological sample, and a heating element for heating the liquid contained in the reaction vessel to a predetermined temperature.

[0060] The term "automated liquid handling system" is used interchangeably with other terms known in the art, such as automated liquid handling / processing / pipetted / dispensing robots / stations / platforms, or similar terms.

[0061] The term "reaction vessel" refers to a space where a reaction for extracting nucleic acids from a sample is performed. The term may be used interchangeably with "container" or "carrier." A reaction vessel accommodates substances such as liquid samples or reagents.

[0062] As used herein, the terms "cell lysis composition" or "composition for lysing cells" refer to a composition that serves to lyse the cells of an organism containing nucleic acids (e.g., DNA or RNA) and release said nucleic acids. Specifically, when the cell lysis composition comes into contact with a biological sample obtained from a subject, it destroys the cells of an organism, such as animals, plants, yeast, bacteria, or viruses, thereby releasing the nucleic acids contained therein to the outside.

[0063] Conventionally, a series of steps including cell lysis, nucleic acid binding, nucleic acid washing, nucleic acid purification, and nucleic acid elution have been performed to isolate nucleic acids from organism cells present in biological samples. Various reagents have been developed to optimize the execution of each step, and these include, but are not limited to, lysis buffers, binding buffers, wash buffers, and elution buffers.

[0064] The “cell lysis composition” of the present disclosure is similar to a conventional lysis buffer in terms of its function of lysing cells. However, since conventional lysis buffers contain components that inhibit nucleic acid amplification reactions such as PCR, the lysate obtained using said buffer cannot be used directly in said reaction without purification. On the other hand, since the cell lysis composition according to the present disclosure does not contain components that inhibit nucleic acid amplification reactions, the lysate obtained using said composition can be used directly in said nucleic acid amplification reaction without purification. In this regard, the “cell lysis composition” according to the present disclosure may also be referred to as a “direct lysis” composition, buffer, reagent, or solution.

[0065] In addition, the “cell lysis composition” according to the present disclosure enables the obtaining of nucleic acids without nucleic acid extraction, i.e., nucleic acid purification, and may also be referred to as an “extraction-free” composition, buffer, reagent, or solution.

[0066] The aforementioned cell lysis composition does not contain an inhibitor of nucleic acid amplification reaction.

[0067] As used herein, "cell lysis" or "lysis" means the breakdown or destruction of an outer boundary or cell membrane to release intracellular substances, such as DNA, RNA, proteins, or organelles, from a cell. In particular, cell lysis or lysis means the breakdown or destruction of a cell to release nucleic acids, namely DNA or RNA, from the cell.

[0068] In the method of the present disclosure, lysis of cells in a biological sample occurs by the cell lysis composition described above.

[0069] Cells in a biological sample that can be dissolved by the composition described above may originate from various organisms, particularly pathogens, such as Gram-positive bacteria.

[0070] In a specific embodiment, the biological sample is Streptococcus piogenes ( Streptococcus pyogenes ), Streptococcus equity subspecies equity ( Streptococcus equi subsp. Equi ) , Streptococcus equi subspecies zueepidemicus ( Streptococcus equi subsp. zooepidemicus ) , Streptococcus disgalactiae subspecies disgalactiae ( Streptococcus dysgalactiae subsp. dysgalactiae ) , Streptococcus disgalactiae subspecies equityimilis ( Streptococcus dysgalactiae subsp. equisimilis ) , Streptococcus cannis ( Streptococcus canis ) , Arcanobacterium hamolitiscum Arcanobacterium haemolyticum It contains or is suspected of containing ), or a combination thereof.

[0071] In a specific embodiment, the cells in the biological sample are Streptococcus piogenes, Streptococcus equi subspecies equi , Streptococcus equi subspecies zueepidemicus , Streptococcus disgalactiae subspecies disgalactiae , Streptococcus disgalactiae subspecies equityimilis , Streptococcus cannis , and It originates from one or more bacteria selected from the group consisting of Arcanobacterium hamolitiscum.

[0072] The cell lysis composition according to the present disclosure is used to isolate nucleic acids from biological samples obtained from subjects who have or are likely to have pharyngitis in order to determine the presence of a causative pathogen in the subjects.

[0073] Pharyngitis is a disease that causes inflammation of the pharynx accompanied by sore throat, and it is transmitted through droplets, coughing, or sneezing. Although pharyngitis itself is not fatal, if not treated with prompt antibiotics, it can lead to various complications such as strep throat and streptococcal toxic shock syndrome. To minimize complications caused by pharyngitis and reduce unnecessary antibiotic use, it is necessary to accurately detect the causative pathogen.

[0074] The main causative pathogens of the above-mentioned pharyngitis are β-hemolytic Streptococcus and Arcanobacterium hamolitiscum ( Arcanobacterium haemolyticum ...etc., and the above example of β-hemolytic streptococcus is Streptococcus piogenes, which belongs to Group A Streptococcus (GAS) ( Streptococcus pyogenes ), and Streptococcus disgalactiae subspecies equityimilis belonging to group C Streptococcus (GCS) or group G Streptococcus (GGS) ( Streptococcus dysgalactiae subsp. equisimilis ) and Streptococcus disgalactiae subspecies disgalactiae( Streptococcus dysgalactiae subsp. dysgalactiae Includes ).

[0075] The above GAS exhibits β-hemolysis and, according to Lancefield's classification, is a Gram-positive bacterium of the genus Streptococcus that has A, C, and G antigens.

[0076] The above GCS or GGS exhibits β-hemolysis and, according to the Lancefield classification, is a Gram-positive bacterium of the genus Streptococcus having C and G antigens.

[0077] Subjects diagnosed with GAS, GCS, or GGS are treated with antibiotics such as penicillin, amoxicillin, or benzathine, and if the subject is allergic to penicillin, they are treated with antibiotics such as cephalexin, cefadroxil, clindamycin, azithromycin, or clarithromycin.

[0078] Meanwhile, Arcanobacterium haemolitisum is a species of bacteria classified as a Gram-positive Bacillus. Individuals diagnosed with Arcanobacterium haemolitisum are treated with antibiotics such as erythromycin, azithromycin, gentamicin, or clindamycin.

[0079] Although prescriptions for GAS, GCS, or GGS differ from those for Arcanobacterium hamolitis, it is difficult to distinguish between them because the symptoms caused by these infections are similar. Therefore, differentiating between them helps prevent the overuse of antibiotics such as penicillin and aids in prescribing accurate treatment.

[0080] The cell lysis composition according to the present disclosure can be used to prepare a sample for a nucleic acid amplification reaction, such as real-time PCR, to detect the causative pathogen in a patient with pharyngitis. Conventionally, performing a nucleic acid amplification reaction requires a complex process of extracting and purifying nucleic acids from biological samples. However, the method according to the present disclosure enables the isolation of nucleic acids from biological samples with only simple incubation, so the lysate obtained by the method of the present disclosure can be applied directly to a nucleic acid amplification reaction.

[0081] In a specific embodiment, the nucleic acid obtained by the method of the present invention can be mixed with a nucleic acid amplification reagent and applied to a nucleic acid amplification reaction.

[0082] As used herein, the term "nucleic acid amplification reagent" refers to a mixture of components used to amplify nucleic acids. The nucleic acid amplification reagent may vary depending on the type of amplification reaction.

[0083] In certain embodiments, nucleic acid amplification reactions include, but are not limited to, PCR, real-time PCR, and LAMP (Loop Mediated Isothermal Amplification).

[0084] Specifically, amplification is carried out according to PCR (polymerase chain reaction), which is disclosed in U.S. Patents No. 4,683,195, 4,683,202 and 4,800,159. Other examples include ligase chain reaction (LCR) (U.S. Patents No. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859 (1995)), and helicase-dependent amplification (helicase dependent amplification (HAD) (M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800), nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991)), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999); Hatch et al., Genet. Anal. 15(2):35-40 (1999)), Q-beta replicase (Lizardi et al.There are , BiolTechnology 6:1197 (1988)), loop-mediated isothermal amplification (LAMP) (Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411) and recombinase polymerase amplification (RPA) (J. Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67).

[0085] In one embodiment, the amplification of the target nucleic acid may be carried out by a nucleic acid amplification method involving a change in temperature, e.g., a PCR method. In such a nucleic acid amplification method, several or tens of cycles including a denaturation step, a primer annealing step, and an extension (or amplification) step may be repeated.

[0086] In one embodiment, the amplification of the target nucleic acid can be carried out by a nucleic acid amplification method that does not involve a change in temperature, i.e., an isothermal amplification method. Examples of isothermal amplification reactions include, without limitation, rolling circle amplification (RCA, MM Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, JA Ankrum, XC Leand W. Zhao, Chem. Soc. Rev., 2014, 43, 3324-3341), loop-mediated isothermal amplification (LAMP, Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), recombinase polymerase amplification (RPA, J. Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67), and nucleic acid sequence-based amplification (NASBA, A. Borst, J. Verhoef, E. Boel and AC Fluit, Clin. Lab., 2002, 48). Includes strand substitution amplification (SDA, BJ Toley, I. Covelli, Y. Belousov, S. Ramachandran, E. Kline, N. Scarr, N. Vermeulen, W. Mahoney, BR Lutz and P. Yager, Analyst, 2015, 140, 7540-7549), helicase-dependent amplification (HAD, M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800), and transcription-mediated amplification (TMA, L. Comanor, Am. J. Gastroenterol., 2001, 96, 2968-2972).

[0087] Although PCR is primarily described throughout the specification as an example of a target nucleic acid amplification method, those skilled in the art will recognize that other nucleic acid amplification reactions may be used in addition to PCR.

[0088] According to one embodiment of the present disclosure, when the target nucleic acid is RNA, the amplification reaction may include a reverse transcription reaction step. Details thereof are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and Noonan, KF et al., Nucleic Acids Res. 16:10366 (1988).

[0089] The nucleic acid amplification reagent used in the method of the present disclosure comprises a set of oligonucleotides for detecting a target nucleic acid.

[0090] The set of oligonucleotides used in the method of the present disclosure comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid; and (ii) a signaling oligonucleotide coupled with a fluorescent label that serves to generate a signal in the presence of the target nucleic acid, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different.

[0091] In one embodiment, the amplified oligonucleotide is a "primer" known in the art. As used herein, the term "primer" refers to an oligonucleotide that can act as a starting point for synthesis when placed under conditions in which the synthesis of a primer extension product complementary to a target nucleic acid (template) is induced, namely, the presence of a polymerizer such as a nucleotide and DNA polymerase, and conditions of suitable temperature and pH. The primer must be sufficiently long to prime the synthesis of the extension product in the presence of a polymerizer. The suitable length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer.

[0092] The primer may include a forward primer (also referred to as an upstream primer or upstream oligonucleotide), a reverse primer (also referred to as a downstream primer or downstream oligonucleotide), or both. The amplified oligonucleotide may be an oligonucleotide having a structure known in the art and may be synthesized in a manner known in the art.

[0093] As used herein, the term "signaling oligonucleotide" refers to an oligonucleotide involved in the generation of a detected signal. According to one embodiment of the present invention, the signaling oligonucleotide comprises an oligonucleotide involved in the actual generation of a signal. For example, hybridization or non-hybridization of the signaling oligonucleotide with another oligonucleotide (e.g., an oligonucleotide comprising a nucleotide sequence complementary to the target nucleic acid or the signaling oligonucleotide) determines the generation of a signal.

[0094] In one embodiment, the signaling oligonucleotide is a “probe” known in the art. As used herein, the term “probe” means a single-stranded nucleic acid molecule comprising a site or sites substantially complementary to the target nucleic acid. According to one embodiment, the 3’-terminus of the probe is “blocked” to prevent its extension. Blocking can be achieved according to conventional methods. For example, blocking can be performed by adding a chemical moiety, such as biotin, a label, a phosphate group, an alkyl group, a non-nucleotide linker, a phosphorothioate, or an alkane-diol residue, to the 3’-hydroxyl group of the last nucleotide. Alternatively, blocking can be performed by removing the 3’-hydroxyl group of the last nucleotide or by using a nucleotide without a 3’-hydroxyl group, such as a dideoxynucleotide.

[0095] In one embodiment, the signaling oligonucleotide comprises at least one fluorescent label, and the at least one fluorescent label within the signaling oligonucleotide generates a fluorescent signal.

[0096] A set of oligonucleotides containing a signaling oligonucleotide can generate a signal by one method known in the art.

[0097] In one embodiment, the set of oligonucleotides generates a signal by the formation of a dimer from a signaling oligonucleotide or by the dissociation of said dimer. In particular, the signal is generated by the formation of a dimer between a target nucleic acid and a signaling oligonucleotide that specifically hybridizes to said target nucleic acid.

[0098] Signals resulting from dimerization between a target nucleic acid and a signaling oligonucleotide are described by the Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), the Sunrise (or Amplifluor) method (Nazarenko et al., Nucleic Acids Research, 25(12):2516-2521 (1997), and U.S. Patent No. 6,117,635), the Lux method (U.S. Patent No. 7,537,886), the Plexor method (Sherrill CB, et al., Journal of the American Chemical Society, 126:4550-4556 (2004)), the Molecular Beacon method (Tyagi et al., Nature Biotechnology v.14 MARCH 1996), the Hybeacon method (French DJ et al., Mol. Cell Probes, 15(6):363-374 (2001)), and adjacent hybridization probes. It can be produced by various methods including the method (Bernard PS et al., Anal. Biochem., 273:221 (1999)) and the LNA method (U.S. Patent No. 6,977,295).

[0099] In one embodiment, the oligonucleotide set generates a signal by the formation or dissociation of a dimer between a signaling oligonucleotide and a cleavage product following the cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs in dependence on the presence of the target nucleic acid.

[0100] The signal by the dimer formed in a manner dependent on the cleavage of the above-mentioned mediating oligonucleotide can be generated by various methods including the PTOCE (PTO cleavage and extension) method (WO 2012 / 096523), the PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013 / 115442), the PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (WO 2014 / 104818), US 11,034,997, US 11,028,433, US 10,590,469, and US 2020-0048682.

[0101] In one embodiment, the oligonucleotide set generates a signal by cleaving the signaling oligonucleotide after hybridization of the signaling oligonucleotide with the target nucleic acid, which occurs dependent on the presence of the target nucleic acid.

[0102] After the signaling oligonucleotide is hybridized to the target nucleic acid, the signal resulting from the cleavage of the signaling oligonucleotide can be generated by various methods including the TaqMan probe method (U.S. Patent No. 5,210,015 and U.S. Patent No. 5,538,848).

[0103] In one embodiment, the set of oligonucleotides generates a signal by the cleavage of a signaling oligonucleotide in a manner dependent on the cleavage of a mediating oligonucleotide specifically hybridized to the target nucleic acid.

[0104] Signals resulting from the cleavage of the aforementioned mediating oligonucleotide-dependent signaling oligonucleotide can be generated by various methods including the Invader analysis (U.S. Patent No. 5,691,142), the PCEC (PTO Cleavage and Extension-Dependent Cleavage) method (WO 2012 / 134195), and the method described in U.S. Patent No. 7,309,573.

[0105] Fluorescent labels useful in the method of the present disclosure are known in the art. For example, fluorescent labels useful in the method of the present disclosure include single fluorescent labels, interactive double fluorescent labels, and interactive triple labels.

[0106] In one embodiment, the single label provides different signal intensities depending on whether it is present on a double strand or a single strand. Preferred types of single fluorescent labels and binding sites used in the method of the present disclosure are disclosed in U.S. Patents No. 7,537,886 and No. 7,348,141, the teachings thereof are incorporated herein by reference in their entirety. For example, the single fluorescent labels include JOE, FAM, TAMRA, ROX, and fluorescein-based labels. The single label may be attached to an oligonucleotide by various methods. For example, the label is attached to a probe through a spacer containing carbon atoms (e.g., a 3-carbon spacer, a 6-carbon spacer, or a 12-carbon spacer).

[0107] II. How to extract nuclear acid

[0108] As described above, the present disclosure provides a method for extracting nucleic acid from a biological sample without purification of nucleic acid by incubating the biological sample with a cell lysis composition.

[0109] In addition, the present disclosure provides a method for extracting nucleic acids in large quantities and conveniently using an automated liquid handling system.

[0110] In addition, the present disclosure provides a method for achieving high nucleic acid extraction efficiency by raising the temperature of the liquid in the reaction vessel to just about 50-60°C.

[0111] Specifically, the present disclosure provides a method for extracting nucleic acids from a biological sample in an automated liquid handling system, comprising the following steps:

[0112] (i) a step of dispensing a cell lysis composition into an empty reaction vessel mounted on a heating element in an automated liquid handling system, wherein the cell lysis composition serves to lyse cells in a biological sample;

[0113] (ii) a step of pre-incubating the cell lysis composition at 50°C or higher using a heating element;

[0114] (iii) a step of preparing a mixture by dispensing a biological sample into the reaction vessel; and

[0115] (iv) a step of extracting nucleic acids from a biological sample by incubating the above mixture at 50°C or higher using a heating element,

[0116] Steps (i)-(iv) are controlled by a controller within an automated liquid handling system, and steps (i) and (iii) are performed by a pipetting channel within the automated liquid handling system.

[0117] The method of the present disclosure is described in detail below.

[0118] Phase (i)

[0119] In step (i), a cell lysis composition is dispensed into an empty reaction vessel mounted on a heating element within an automated liquid handling system.

[0120] Step (i) above is controlled by a controller within the automated liquid handling system. Additionally, Step (i) is performed by a pipetting channel within the automated liquid handling system.

[0121] Descriptions of the above automated liquid handling systems are found elsewhere in this application. Examples of automated liquid handling systems available in the art include, but are not limited to, Hamilton’s Microlab STARlet, Microlab STAR, and Microlab NIMBUS; and Seegene’s Seegene STARlet or STARlet-AIOS.

[0122] The aforementioned heating element is provided within an automated liquid handling system.

[0123] As used herein, the term "heating element" refers to a device that serves to raise the temperature of a contacted object. In this invention, the heating element serves to raise the temperature of a reaction vessel placed thereon or a substance within the reaction vessel, such as a cell lysis composition or a mixture of a cell lysis composition and a biological sample.

[0124] In one embodiment, the heating element functions to heat a reaction vessel mounted thereon to a predetermined temperature.

[0125] In one embodiment, the heating element functions to heat to a predetermined temperature while shaking the reaction vessel mounted thereon. The term "heating element" may be used interchangeably with various terms known in the art, such as heating unit, heating module, heating component, heating element, heating means, heater, heater shaker, etc.

[0126] The heating element may have a size that can cover the entire reaction vessel. As an example, the heating element may have a length of 100-200 mm, a width of 50-150 mm, and a height of 70-150 mm, and more particularly, a length of 150 mm, a width of 105 mm, and a height of 90 mm.

[0127] The heating element may have a heating range from room temperature (e.g., 25°C) to over 100°C. As an example, the heating element may heat to a predetermined temperature of 105°C.

[0128] However, there may be a significant difference between the predetermined temperature of the heating element and the temperature of the liquid in the reaction vessel. This is because it is difficult for the liquid in the reaction vessel to rise to the predetermined temperature of the heating element due to various factors, such as the low thermal conductivity of the reaction vessel or the liquid in the reaction vessel. For example, even if the heating element is intended to rise to a very high temperature, such as 100°C, the liquid in the deep-well plate serving as the reaction vessel may only be at a temperature of 55°C to 60°C.

[0129] Although the predetermined temperature of the heating element and the temperature of the liquid in the reaction vessel, such as a cell lysis composition or a mixture of said composition and a biological sample, are described in the specification, it should be noted that the temperature of said composition or the mixture of said composition and a biological sample is an important factor in achieving the purpose of the present disclosure. The heating element of the present disclosure is freely adjusted to achieve the target temperature of the composition or mixture. For example, the predetermined temperature of the heating element for raising a liquid in a reaction vessel with low thermal conductivity to a specific temperature will be higher than the predetermined temperature of the heating element for raising a liquid in a reaction vessel with high thermal conductivity to the same temperature. A person skilled in the art will be able to easily determine the predetermined temperature of the heating element for raising the liquid in said reaction vessel to a specific temperature depending on the type of reaction vessel.

[0130] The heating element according to the present disclosure can shake the reaction vessel at various speeds. As an example, the heating element can shake the reaction vessel at a speed of 200 to 3000 rpm, for example, 2500 rpm.

[0131] The heating element according to the present disclosure may include an adapter as an auxiliary means for mounting a reaction vessel. The adapter may vary to suit the reaction vessel to be mounted.

[0132] In addition, the heating element according to the present disclosure may further include a cooling means for lowering the temperature.

[0133] Examples of heating elements available in the industry include, but are not limited to, Hamilton's heater shakers.

[0134] A reaction vessel according to the present disclosure is mounted on a heating element within an automated liquid handling system.

[0135] The above reaction vessel is an empty reaction vessel that is not filled with liquid.

[0136] The reaction vessel used in the method of the present disclosure is not particularly limited and may be, for example, a deep-well plate or a well plate.

[0137] Specific examples of deep-well plates include 1.2 mL deep-well plates and 2.2 mL deep-well plates, and specific examples of well plates include 96-well plates and 384-well plates.

[0138] In a specific embodiment, the reaction vessel according to the present disclosure is a deep-well plate.

[0139] In one embodiment, the deep well plate is made of polypropylene and has a length of about 127.63-127.80 mm, a height of about 41.6-44.4 mm, and a depth of about 84.63-85.5 mm.

[0140] The deep well plates mentioned above may include deep well plates manufactured by various manufacturers, such as Hamilton, Thermo Fisher Scientific, Axygen, Corning, etc., but are not limited thereto.

[0141] As described above, the cell lysis composition dispensed into the reaction vessel in step (i) may be a direct lysis buffer or a non-extraction buffer known in the art. As described elsewhere in this application, since the cell lysis composition is used to obtain nucleic acids to be applied directly to a subsequent reaction without further purification, it must not contain components that inhibit a subsequent amplification reaction, such as real-time PCR.

[0142] In a specific embodiment, the cell lysis composition is

[0143] (a) Guanidine hydrochloride (GuHCl);

[0144] (b) cetyltrimethylammonium bromide (CTAB); and

[0145] (c) Polyethylene glycol (PEG) having a molecular weight of 200 to 1000 Da

[0146] Includes

[0147] The cell lysis composition according to the present disclosure has a combination of components and their optimal concentrations that have an excellent cell lysis effect without inhibiting nucleic acid amplification reactions.

[0148] Hereinafter, each component included in the composition of the present invention and their optimal concentrations will be described.

[0149] guanidine hydrochlorothiazide (GuHCl)

[0150] Guanidine hydrochloride (GuHCl), also known as guanidinium chloride (GdmCl), is a strong chaotrope capable of disrupting the hydrogen bond network between water molecules. GuHCl is one of the strongest denaturants used in the biochemical study of protein folding. It also possesses the ability to reduce enzyme activity and increase the solubility of hydrophobic molecules.

[0151] In the method according to the present disclosure, GuHCl plays the role of denaturing proteins and lysing cells.

[0152] The above GuHCl may be included in the composition in an amount of about 30 to about 300 mM. According to an example of the present disclosure, 500 mM or more of GuHCl exhibits inhibitory activity against nucleic acid amplification reactions.

[0153] In one embodiment, the GuHCl is included in the composition in an amount of 30 to 100 mM, 30 to 150 mM, 30 to 200 mM, 30 to 250 mM, 30 to 300 mM, 50 to 100 mM, 50 to 150 mM, 50 to 200 mM, 50 to 250 mM, 50 to 300 mM, 100 to 150 mM, 100 to 200 mM, 100 to 250 mM, 100 to 300 mM, 150 to 200 mM, 150 to 250 mM, 150 to 300 mM, 200 to 250 mM, 200 to 300 mM, 250 to 300 mM, or any value in between.

[0154] In a specific embodiment, the GuHCl is included in the composition in an amount of about 100 mM.

[0155] The above GuHCl may be included in an amount of about 0.3 to about 3 weight percent based on the total weight of the composition.

[0156] In one embodiment, the GuHCl is present in an amount of 0.3 to 1.0 wt%, 0.3 to 1.5 wt%, 0.3 to 2.0 wt%, 0.3 to 2.5 wt%, 0.3 to 3.0 wt%, 0.5 to 1.0 wt%, 0.5 to 1.5 wt%, 0.5 to 2.0 wt%, 0.5 to 2.5 wt%, 0.5 to 3.0 wt%, 1.0 to 1.5 wt%, 1.0 to 2.0 wt%, 1.0 to 2.5 wt%, 1.0 to 3.0 wt%, 1.5 to 2.0 wt%, 1.5 to 2.5 wt%, 1.5 to 3.0 wt%, 2.0 to 2.5 wt%, 2.0 to 3.0 wt%, based on the total weight of the composition. It is included in an amount of 2.5 to 3.0 weight%, or any value in between.

[0157] In a specific embodiment, the GuHCl is included in an amount of 1.0 weight% based on the total weight of the composition.

[0158] A lysate obtained using a composition containing a combination of CTAB and PEG described below exhibits a reduced fluorescence signal, i.e., a reduced RFU value, in a nucleic acid amplification reaction, whereas adding GuHCl to the composition containing the combination of CTAB and PEG can prevent the reduction of the fluorescence signal.

[0159] methyltrimethylammonium bromide (CTAB)

[0160] Cetyltrimethylammonium bromide (CTAB), also known as hexadecyltrimethylammonium bromide, is [(C 16 H 33 It has the chemical formula )N(CH3)3]Br. CTAB is a cationic surfactant, and the positive charge of CTAB electrostatically binds to the negatively charged teichoic acid present in Gram-positive cells, causing stress to the cells and lysing them.

[0161] CTAB plays a role in maintaining the integrity of DNA that precipitates during nucleic acid isolation. Cells typically contain high concentrations of macromolecules, such as glycoproteins and polysaccharides, which precipitate along with DNA during the extraction process, thereby lowering the purity of the extracted DNA. The positive charge of CTAB helps increase DNA purity by denaturing these molecules that would otherwise interfere with nucleic acid isolation.

[0162] The above CTAB may be included in an amount of about 0.05 to less than about 0.5 weight% based on the total weight of the composition. According to the embodiments of the present disclosure, 0.5 weight% or more of CTAB exhibits inhibitory activity against nucleic acid amplification reactions.

[0163] In one embodiment, the CTAB is included in an amount of 0.05 to 0.1 wt%, 0.05 to 0.2 wt%, 0.05 to 0.3 wt%, 0.05 to 0.4 wt%, 0.1 to 0.2 wt%, 0.1 to 0.3 wt%, 0.1 to 0.4 wt%, 0.15 to 0.2 wt%, 0.15 to 0.3 wt%, 0.15 to 0.4 wt%, 0.2 to 0.3 wt%, 0.2 to 0.4 wt%, 0.25 to 0.3 wt%, 0.25 to 0.4 wt%, 0.3 to 0.4 wt%, 0.35 to 0.4 wt%, or any value between these, based on the total weight of the composition.

[0164] In a specific embodiment, the CTAB is included in an amount of about 0.1 weight% based on the total weight of the composition.

[0165] Lysates obtained using compositions containing CTAB exhibit low Ct values, i.e., high performance, in nucleic acid amplification reactions.

[0166] polyethylene glycol (PEG)

[0167] Polyethylene glycol (PEG) is H(OCH2CH2) n It has the chemical formula OH and is produced by the polymerization of ethylene oxide. PEG is a hydrophilic compound that acts as a cathode by binding to water molecules, thereby lysing cells.

[0168] The above PEG may be included in an amount of about 5 to less than about 80 weight percent based on the total weight of the composition. According to the embodiments of the present disclosure, PEG does not exhibit inhibitory activity against nucleic acid amplification reactions even when added at high concentrations.

[0169] In one embodiment, the PEG is 5 to 10 wt%, 5 to 20 wt%, 5 to 30 wt%, 5 to 40 wt%, 5 to 50 wt%, 5 to 60 wt%, 5 to 70 wt%, 5 to 80 wt%, 10 to 20 wt%, 10 to 30 wt%, 10 to 40 wt%, 10 to 50 wt%, 10 to 60 wt%, 10 to 70 wt%, 10 to 80 wt%, 20 to 30 wt%, 20 to 40 wt%, 20 to 50 wt%, 20 to 60 wt%, 20 to 70 wt%, 20 to 80 wt%, 30 to 40 wt%, 30 to 50 wt%, 30 to 60 wt%, based on the total weight of the composition. It is included in an amount of weight%, 30 to 70 weight%, 30 to 80 weight%, 40 to 50 weight%, 40 to 60 weight%, 40 to 70 weight%, 40 to 80 weight%, 50 to 60 weight%, 50 to 70 weight%, 50 to 80 weight%, 60 to 70 weight%, 60 to 80 weight%, 70 to 80 weight%, or any value in between.

[0170] In a specific embodiment, the PEG is included in an amount of 60 weight percent based on the total weight of the composition.

[0171] The above PEG may have various molecular weights known in the art, for example, a molecular weight of 300 to 10,000,000 Da (g / mol).

[0172] In a specific embodiment, the PEG has a molecular weight of 200 to 1,000 Da.

[0173] In one embodiment, the PEG is PEG200. The PEG200 has an average molecular weight of about 190 to about 210 Da and is a colorless, transparent liquid.

[0174] In one embodiment, the PEG is PEG300. The PEG300 has an average molecular weight of about 290 to about 305 Da and is a nearly colorless, transparent liquid.

[0175] In one embodiment, the PEG is PEG400. The PEG400 has an average molecular weight of about 380 to about 420 Da and is a colorless, transparent liquid.

[0176] In one embodiment, the PEG is PEG600. The PEG600 has an average molecular weight of about 550 to about 650 Da and is a nearly white or colorless powder.

[0177] In one embodiment, the PEG is PEG900. The PEG900 has an average molecular weight of about 850 to about 950 Da and is a nearly white or colorless powder.

[0178] In one embodiment, the PEG is PEG1000. The PEG1000 has an average molecular weight of about 900 to about 1,100 Da and is a nearly white or colorless powder.

[0179] The lysate obtained using a composition containing PEG exhibits low Ct values ​​and high RFU values, i.e., high performance, in nucleic acid amplification reactions.

[0181] As described above, the cell lysis composition according to the present disclosure comprises a combination of GuHCl, CTAB, and PEG.

[0182] Isolation and amplification of nucleic acids using a cell lysis composition containing CTAB and PEG exhibit a reduced Ct value compared to conventional nucleic acid extraction and amplification, i.e., improved detection performance of target nucleic acids.

[0183] In addition, the isolation and amplification of nucleic acids using a cell lysis composition containing a combination of GuHCl, CTAB, and PEG exhibits higher RFU values, i.e., improved detection performance of target nucleic acids, compared to the isolation and amplification of nucleic acids using a cell lysis composition containing CTAB and PEG.

[0184] Therefore, a cell lysis composition containing a combination of GuHCl, CTAB, and PEG can be used to detect target nucleic acids with higher RFU values ​​and lower Ct values.

[0185] In a specific embodiment, the cell lysis composition comprises an amount of 30 to 300 mM of GuHCl, an amount of less than 0.05 to 0.5 weight% of CTAB based on the total weight of the cell lysis composition, and an amount of 5 to 80 weight% of PEG based on the total weight of the cell lysis composition.

[0186] In a specific embodiment, the cell lysis composition according to the present disclosure is

[0187] (a) Approximately 100 mM of GuHCl;

[0188] (b) about 0.1 wt% of CTAB; and

[0189] (c) Approximately 60 wt% PEG200

[0190] Includes

[0191] detergent

[0192] The cell lysis composition according to the present disclosure may further include a cationic, anionic, or nonionic detergent.

[0193] In one embodiment, the cationic detergent is selected from the group consisting of benzalkonium chloride (BAC), benzethonium chloride (BEC), methylbenzethonium, and cetylpyridinium chloride.

[0194] In one embodiment, the anionic detergent is n-lauroylsarcosine (NLS).

[0195] NLS, also known as sodium lauroyl sarcosinate or N-dodecanoyl-N-methylglycine sodium salt, is CH3(CH2) 10 It has the chemical formula CON(CH3)CH2COONa. NLS acts on cell membranes and proteins to lyse cells.

[0196] The above NLS may be included in an amount of about 0.05 to less than about 1 weight percent based on the total weight of the composition. According to an example of the present disclosure, 1 weight percent or more of NLS exhibits inhibitory activity against nucleic acid amplification reactions.

[0197] In one embodiment, the NLS is present in an amount of 0.05 to 0.9 wt%, 0.05 to 0.8 wt%, 0.05 to 0.7 wt%, 0.05 to 0.6 wt%, 0.05 to 0.5 wt%, 0.05 to 0.4 wt%, 0.05 to 0.3 wt%, 0.05 to 0.2 wt%, 0.05 to 0.1 wt%, 0.1 to 0.9 wt%, 0.1 to 0.8 wt%, 0.1 to 0.7 wt%, 0.1 to 0.6 wt%, 0.1 to 0.5 wt%, 0.1 to 0.4 wt%, 0.1 to 0.3 wt%, 0.1 to 0.2 wt%, 0.2 to 0.9 wt%, 0.2 to based on the total weight of the composition. 0.8 wt%, 0.2 to 0.7 wt%, 0.2 to 0.6 wt%, 0.2 to 0.5 wt%, 0.2 to 0.4 wt%, 0.2 to 0.3 wt%, 0.3 to 0.9 wt%, 0.3 to 0.8 wt%, 0.3 to 0.7 wt%, 0.3 to 0.6 wt%, 0.3 to 0.5 wt%, 0.3 to 0.4 wt%, 0.4 to 0.9 wt%, 0.4 to 0.8 wt%, 0.4 to 0.7 wt%, 0.4 to 0.6 wt%, 0.4 to 0.5 wt%, 0.5 to 0.9 wt%, 0.5 to 0.8 wt%, 0.5 to 0.7 wt%, 0.5 to 0.6 wt%, 0.5 to 0.6 wt%, 0.5 to 0.8 wt%, 0.5 to 0.7 wt%, 0.5 to 0.6 wt% It is included in an amount of weight%, 0.6 to 0.9 weight%, 0.6 to 0.8 weight%, 0.6 to 0.7 weight%, 0.7 to 0.9 weight%, 0.7 to 0.8 weight%, 0.8 to 0.9 weight%, or any value in between.

[0198] In a specific embodiment, the NLS is included in an amount of 0.05 weight% based on the total weight of the composition.

[0199] In one embodiment, the nonionic detergent comprises polyoxyethylene sorbitan monolaurate (Tween 20).

[0200] Tween 20, also known as polysorbate 20 or PEG (20) sorbitan monolaurate, is C 58 H 114 O 26 It has the chemical formula. Tween 20 acts on cell membranes and proteins to lyse cells.

[0201] The above Tween 20 may be included in an amount of about 5 to about 15 weight percent based on the total weight of the composition. According to an example of the present disclosure, 20 weight percent or more of Tween 20 exhibits inhibitory activity against nucleic acid amplification reactions.

[0202] In one embodiment, the Tween 20 is 5 to 6 wt%, 5 to 7 wt%, 5 to 8 wt%, 5 to 9 wt%, 5 to 10 wt%, 5 to 11 wt%, 5 to 12 wt%, 5 to 13 wt%, 5 to 14 wt%, 5 to 15 wt%, 6 to 7 wt%, 5 to 8 wt%, 6 to 9 wt%, 6 to 10 wt%, 6 to 11 wt%, 6 to 12 wt%, 6 to 13 wt%, 6 to 14 wt%, 6 to 15 wt%, 7 to 8 wt%, 7 to 9 wt%, 7 to 10 wt%, 7 to 11 wt%, 7 to 12 wt%, 7 to 13 wt%, 7 to 14 wt%, 7 to 14 wt%, 7 based on the total weight of the composition. to 15 wt%, 8 to 9 wt%, 8 to 10 wt%, 8 to 11 wt%, 8 to 12 wt%, 8 to 13 wt%, 8 to 14 wt%, 8 to 15 wt%, 9 to 10 wt%, 9 to 11 wt%, 9 to 12 wt%, 9 to 13 wt%, 9 to 14 wt%, 9 to 15 wt%, 10 to 11 wt%, 10 to 12 wt%, 10 to 13 wt%, 10 to 14 wt%, 10 to 15 wt%, 11 to 12 wt%, 11 to 13 wt%, 11 to 14 wt%, 11 to 15 wt%, 11 to 15 wt%, 12 to 13 wt%, 12 to 14 wt%, 12 to 15 wt%, 13 It is included in an amount of up to 14 weight%, 13 to 15 weight%, 14 to 15 weight%, or any value in between.

[0203] In a specific embodiment, the Tween 20 is included in an amount of 5% by weight based on the total weight of the composition.

[0204] Chelating agent

[0205] The cell lysis composition according to the present disclosure may further include a chelating agent.

[0206] In one embodiment, the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0207] EDTA is a metal chelating agent that chelates divalent cations, such as magnesium, zinc, manganese, nickel, or copper ions, which are cofactors of many enzymes, such as DNAases and proteases. By chelating these enzyme cofactors, EDTA inactivates these enzymes, thereby preventing the degradation of nucleic acids. Additionally, EDTA facilitates the dissociation of 40S and 60S ribosomal subunits.

[0208] The above EDTA may be included in the composition in an amount of about 0.1 to about 10 mM.

[0209] In one embodiment, the EDTA is 0.1 to 1 mM, 0.1 to 2 mM, 0.1 to 3 mM, 0.1 to 4 mM, 0.1 to 5 mM, 0.1 to 6 mM, 0.1 to 7 mM, 0.1 to 8 mM, 0.1 to 9 mM, 0.1 to 10 mM, 1 to 2 mM, 1 to 3 mM, 1 to 4 mM, 1 to 5 mM, 1 to 6 mM, 1 to 7 mM, 1 to 8 mM, 1 to 9 mM, 1 to 10 mM, 2 to 3 mM, 2 to 4 mM, 2 to 5 mM, 2 to 6 mM, 2 to 7 mM, 2 to 8 mM, 2 to 9 mM, 2 to 10 mM, 3 to 4 mM, 3 to 5 mM, 3 to 6 mM, 3 to It is included in an amount of 7 mM, 3 to 8 mM, 3 to 9 mM, 3 to 10 mM, 4 to 5 mM, 4 to 6 mM, 4 to 7 mM, 4 to 8 mM, 4 to 9 mM, 4 to 10 mM, 5 to 6 mM, 5 to 7 mM, 5 to 8 mM, 5 to 9 mM, 5 to 10 mM, 6 to 7 mM, 6 to 8 mM, 6 to 9 mM, 6 to 10 mM, 7 to 8 mM, 7 to 9 mM, 7 to 10 mM, 8 to 9 mM, 8 to 10 mM, 9 to 10 mM, or any value in between.

[0210] In a specific embodiment, the EDTA is included in the composition in an amount of about 1 mM.

[0211] The above EDTA may be included in an amount of about 0.02 to about 2 weight percent based on the total weight of the composition.

[0212] In one embodiment, the EDTA is present in an amount of 0.02 to 0.2 wt%, 0.02 to 0.4 wt%, 0.02 to 0.6 wt%, 0.02 to 0.8 wt%, 0.02 to 1.0 wt%, 0.02 to 1.2 wt%, 0.02 to 1.4 wt%, 0.02 to 1.6 wt%, 0.02 to 1.8 wt%, 0.02 to 2 wt%, 0.1 to 0.2 wt%, 0.1 to 0.4 wt%, 0.1 to 0.6 wt%, 0.1 to 0.8 wt%, 0.1 to 1.0 wt%, 0.1 to 1.2 wt%, 0.1 to 1.4 wt%, 0.1 to 1.6 wt%, 0.1 to 1.8 wt%, 0.1 to 2.0 wt%, 0.2 to 0.4 wt%, 0.2 to 0.6 wt%, 0.2 to 0.8 wt%, 0.2 to 1.0 wt%, 0.2 to 1.2 wt%, 0.2 to 1.4 wt%, 0.2 to 1.6 wt%, 0.2 to 1.8 wt%, 0.2 to 2.0 wt%, 0.4 to 0.6 wt%, 0.4 to 0.8 wt%, 0.4 to 1.0 wt%, 0.4 to 1.2 wt%, 0.4 to 1.4 wt%, 0.4 to 1.6 wt%, 0.4 to 1.8 wt%, 0.4 to 2.0 wt%, 0.6 to 0.8 wt%, 0.6 to 1.0 wt%, 0.6 to 1.2 wt%, 0.6 to 1.4 wt%, 0.6 to 1.6 wt%, 0.6 to 1.8 wt%, 0.6 to 2.0 wt%, 0.8 to 1.0 wt%, 0.8 to 1.2 wt%, 0.8 to 1.4 wt%, 0.8 to 1.6 wt%, 0.8 to 1.8 wt%, 0.8 to 2.0 wt%, 1.0 to 1.2 wt%, 1.0 to 1.4 wt%, 1.0 to 1.6 wt%, 1.0 to 1.8 wt%, 1.0 to 2.0 wt%, 1.2 to 1.4 wt%, 1.2 to 1.6 wt%, 1.2 to 1.8 wt%, 1.2 to 2.0 wt%, 1.4 to 1.It is included in an amount of 6 wt%, 1.4 to 1.8 wt%, 1.4 to 2 wt%, 1.6 to 1.8 wt%, 1.6 to 2 wt%, 1.8 to 2 wt%, or any value in between.

[0213] buffer

[0214] The cell lysis composition according to the present disclosure may further include a buffer.

[0215] In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris).

[0216] Tris, also known as trisamine, prometamol, tribase, Trizma, or THAM, is a primary amine with the chemical formula (HOCH2)3CNH2. Tris plays a role in preventing rapid changes in pH by maintaining a constant hydrogen ion concentration.

[0217] The above Tris may be included in the composition in an amount of about 1 to about 50 mM.

[0218] In one embodiment, the Tris is 1 to 5 mM, 1 to 10 mM, 1 to 15 mM, 1 to 20 mM, 1 to 25 mM, 1 to 30 mM, 1 to 35 mM, 1 to 40 mM, 1 to 45 mM, 1 to 50 mM, 5 to 10 mM, 5 to 15 mM, 5 to 20 mM, 5 to 25 mM, 5 to 30 mM, 5 to 35 mM, 5 to 40 mM, 5 to 45 mM, 5 to 50 mM, 10 to 15 mM, 10 to 20 mM, 10 to 25 mM, 10 to 30 mM, 10 to 35 mM, 10 to 40 mM, 10 to 45 mM, 10 to 50 mM, 15 to 20 mM, 15 to 25 mM, 15 to 30 mM, 15 to 35 mM, 15 to 40 mM, 15 to 45 mM, 15 to 50 mM, 20 to 25 mM, 20 to 30 mM, 20 to 35 mM, 20 to 40 mM, 20 to 45 mM, 20 to 50 mM, 25 to 30 mM, 25 to 35 mM, 25 to 40 mM, 25 to 45 mM, 25 to 50 mM, 30 to 35 mM, 30 to 40 mM, 30 to 45 mM, 30 to 50 mM, 35 to 40 mM, 35 to 45 mM, 35 to 50 mM, 40 to 45 mM, 40 to 50 mM, 45 to 50 mM, or any value in between. It is included in the amount.

[0219] In a specific embodiment, the Tris is included in the composition in an amount of about 10 mM.

[0220] The above Tris may be included in an amount of about 0.01 to about 0.5 weight percent based on the total weight of the composition.

[0221] In one embodiment, the Tris is 0.01 to 0.05 wt%, 0.01 to 0.10 wt%, 0.01 to 0.15 wt%, 0.01 to 0.20 wt%, 0.01 to 0.25 wt%, 0.01 to 0.30 wt%, 0.01 to 0.35 wt%, 0.01 to 0.40 wt%, 0.01 to 0.45 wt%, 0.01 to 0.50 wt%, 0.05 to 0.10 wt%, 0.05 to 0.15 wt%, 0.05 to 0.20 wt%, 0.05 to 0.25 wt%, 0.05 to 0.30 wt%, 0.05 to 0.35 wt%, 0.05 to 0.40 wt%, 0.05 to 0.45 wt%, 0.05 to 0.50 wt%, 0.10 to 0.15 wt%, 0.10 to 0.20 wt%, 0.10 to 0.25 wt%, 0.10 to 0.30 wt%, 0.10 to 0.35 wt%, 0.10 to 0.40 wt%, 0.10 to 0.45 wt%, 0.10 to 0.50 wt%, 0.15 to 0.20 wt%, 0.15 to 0.25 wt%, 0.15 to 0.30 wt%, 0.15 to 0.35 wt%, 0.15 to 0.40 wt%, 0.15 to 0.45 wt%, 0.15 to 0.50 wt%, 0.20 to 0.25 wt%, 0.20 to 0.30 wt%, 0.20 to 0.35 wt%, 0.20 to 0.40 wt%, 0.20 to 0.45 wt%, 0.20 to 0.50 wt%, 0.25 to 0.30 wt%, 0.25 to 0.35 wt%, 0.25 to 0.40 wt%, 0.25 to 0.45 wt%, 0.25 to 0.50 wt%, 0.30 to 0.35 wt%, 0.30 to 0.40 wt%, 0.30 to 0.45 wt%, 0.30 to 0.50 wt%, 0.35 to 0.40 wt%, 0.35 to 0.45 wt%, 0.35 to 0.45 wt%, 0.35 to 0.50 wt%, 0.40 to 0.45 wt%, 0.40 to 0.It is included in an amount of 50 weight%, 0.45 to 0.50 weight%, or any value in between.

[0222] In a specific embodiment, the Tris is included in an amount of 0.1 weight% based on the total weight of the composition.

[0223] The cell lysis composition according to the present disclosure has a pH of about 8 or higher. The pH of the cell lysis composition does not significantly affect the cell lysis and nucleic acid amplification reactions. Those skilled in the art may appropriately adjust the pH of the cell lysis composition as long as the cell lysis composition does not significantly affect the cell lysis and nucleic acid amplification reactions. In one embodiment, the cell lysis composition has a pH of about 8. In another embodiment, the cell lysis composition has a pH of 8 to 12, for example, pH 8, pH 9, pH 10, pH 11, or pH 12.

[0224] In a specific embodiment, the cell lysis composition according to the present disclosure is

[0225] (a) Approximately 100 mM of GuHCl;

[0226] (b) about 0.1 wt% of CTAB;

[0227] (c) Approximately 60 wt% of PEG200;

[0228] (d) About 0.05 wt% of NLS;

[0229] (e) About 5 wt% of Tween 20;

[0230] (f) about 1 mM EDTA; and

[0231] (g) Approximately 10 mM of Tris

[0232] It includes, wherein the composition has a pH of about 8.

[0233] Those skilled in the art will understand that similar performance can be achieved by slightly changing the optimal concentrations of each component constituting the cell lysis composition.

[0234] In one embodiment, the cell lysis composition described above in step (i) is dispensed in an amount of 10 to 100 μL. In a specific embodiment, the cell lysis composition is dispensed in an amount of 30 μL, 40 μL, 50 μL, 60 μL, or 70 μL. In a specific embodiment, the cell lysis composition is dispensed in an amount of 50 μL. The dispensed amount is per reaction vessel, and, for example, if the reaction vessel is a 96-deep-well plate, 50 μL of the cell lysis composition may be dispensed into each well.

[0235] In one embodiment, the method according to the present disclosure further comprises the step of heating an empty reaction vessel by a heating element in an automated liquid handling system prior to step (i). Preheating the empty reaction vessel as described above can help to rapidly increase the temperature of the cell lysis composition filled into the empty reaction vessel in step (i).

[0236] In one embodiment, the heating element is controlled by a controller so that its temperature increases to 95°C or higher. In a specific embodiment, the temperature of the heating element may be heated to a predetermined temperature, e.g., 95°C, 100°C, or 105°C.

[0237] The time for heating is not significantly limited and, for example, may be 1 to 10 minutes, specifically 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0238] The heating can be performed as soon as the empty reaction vessel is mounted on the heating element, or as soon as the power to the automated liquid handling system is turned on.

[0239] Step (ii): Pre-incubation of the cell lysis composition

[0240] In step (ii), the cell lysis composition is pre-incubated at a temperature of about 50°C or higher by a heating element.

[0241] Step (ii) is controlled by a controller within an automated liquid handling system.

[0242] According to an embodiment of the present disclosure, it has been confirmed that there are limitations to heating the liquid in a reaction vessel using a heating element within an automated liquid handling system. Specifically, it has been confirmed that raising the heating element to a very high temperature, e.g., 100-105°C, cannot raise the temperature of the liquid in a reaction vessel, such as a deep-well plate, e.g., a cell lysis composition, to more than 55-60°C. When attempting to extract nucleic acids without purification using a cell lysis composition as in the method of the present disclosure, it is impossible to heat the mixture of the cell lysis composition and the biological sample to approximately 100°C.

[0243] The inventors have discovered that pre-incubating, for example, pre-heating the cell lysis composition before mixing it with a biological sample results in improved extraction efficiency compared to pre-incubating, for example, pre-heating only the biological sample.

[0244] Pre-incubation of biological samples results in the evaporation of liquid components within the biological samples and the subsequent concentration of PCR inhibitors (if present), whereas pre-incubation of cell lysis compositions is not expected to result in the concentration of PCR inhibitors due to the high boiling points of most chemicals constituting the cell lysis compositions, e.g., above 100°C.

[0245] In one embodiment, the cell lysis composition is incubated at about 55°C to 65°C. To this end, the heating element may be controlled by a controller so that its temperature increases to 95°C or higher. The increase in the temperature of the heating element to 95°C or higher may maintain the temperature of the cell lysis composition in the reaction vessel at a temperature of 55°C to 65°C.

[0246] In a specific embodiment, the heating element may be controlled by a controller so that its temperature increases to 100°C. In a specific embodiment, the heating element may be controlled by a controller so that its temperature increases to 105°C.

[0247] In one embodiment, the pre-incubation in step (ii) is performed for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, or a time in between. In a specific embodiment, the pre-incubation in step (ii) is performed for about 10 minutes.

[0248] The pre-incubation time affects the efficiency and sensitivity of the subsequent nucleic acid amplification reaction. Considering the improvement in the efficiency and sensitivity of the nucleic acid amplification reaction, the optimal pre-incubation time of the cell lysis composition is about 10 minutes.

[0249] Step (iii): Dispensing of biological samples

[0250] In step (iii), a biological sample is dispensed into the reaction vessel to prepare a mixture.

[0251] Step (iii) is controlled by a controller within the automated liquid handling system. Additionally, step (iii) is performed by a pipetting channel within the automated liquid handling system.

[0252] Prior to step (iii), the pre-incubated cell lysis composition is mixed with the biological sample. The mixing lyses the cells within the biological sample, thereby releasing nucleic acids present within the cells to the outside of the cells.

[0253] The above mixing may be performed, for example, by (i) immersing a biological sample collected from a subject in a transport medium and then mixing a portion of the transport medium with the aforementioned cell lysis composition, or (ii) directly mixing the biological sample with the cell lysis composition.

[0254] When mixing a biological sample immersed in a transport medium with a cell lysis composition, the collected biological sample is immersed in the transport medium, and a portion of the transport medium containing the biological sample may be mixed with the cell lysis composition. The transport medium refers to a medium capable of maintaining the integrity of the sample, for example, the cellular integrity of viruses or bacteria.

[0255] Examples of commercially available transport media include, but are not limited to, universal transport medium (UTM) from Copan Diagnostics Inc., viral transport medium (VTM) from Asan Pharm. Co., Ltd., Clinical Virus Transport Medium (CTM) from Noble Biosciences, Inc., and ALLTM medium from SG medical Inc.

[0256] The transport medium may be a saline-based solution or a balanced salt solution-based medium. As a specific example, the saline-based solution may be PBS (phosphate buffered saline) or normal saline.

[0257] The transport medium may not contain cell lysis components. Additionally, the transport medium may be a liquid medium.

[0258] According to one embodiment, a transport medium containing a cell lysis composition may be mixed with a biological sample in a volume ratio of 3:1 to 1:3, 5:2 to 2:5, 2:1 to 1:2, 3:2 to 2:3, 4:3 to 3:4, 5:4 to 4:5, or about 1:1. Mixing them in the above volume ratios can improve the performance of nucleic acid isolation using the cell lysis composition and the amplification of the isolated nucleic acid.

[0259] In one embodiment, the biological sample of step (iii) may be dispensed in an amount of 10 to 100 μL. In a specific embodiment, the biological sample of step (iii) is dispensed in an amount of 30 μL, 40 μL, 50 μL, 60 μL, or 70 μL. In a specific embodiment, the biological sample of step (iii) is dispensed in an amount of 50 μL. The dispensed amount is per reaction vessel, and, for example, if the reaction vessel is a 96-deep-well plate, 50 μL of the biological sample may be dispensed into each well.

[0260] In one embodiment, the biological sample of step (iii) is dispensed in an equal amount to the cell lysis composition dispensed in step (i).

[0261] In a specific embodiment, the cell lysis composition is dispensed in an amount of 50 μL in step (i), and the biological sample is also dispensed in an amount of 50 μL in step (iii).

[0262] When a biological sample is mixed directly with a cell lysis composition, the collected sample may be immersed in the aforementioned cell lysis composition.

[0263] Step (iv): Incubation of the mixture

[0264] In step (iv), the mixture is incubated at 50°C or higher by a heating element to extract nucleic acids from the biological sample.

[0265] Step (iv) is controlled by a controller within the automated liquid handling system.

[0266] Incubation in step (iv) weakens the cells within the biological sample to induce an environment where nucleic acids are easily released outside the cells, and induces the chemicals in the cell lysis composition to react actively with the cells to lyse them.

[0267] In one embodiment, the mixture may be incubated at about 55°C to 65°C. To this end, the heating element may be controlled by a controller so that its temperature increases to 95°C or higher. Increasing the temperature of the heating element to 95°C or higher may maintain the temperature of the mixture in the reaction vessel at 55°C to 65°C.

[0268] In a specific embodiment, the heating element may be controlled by a controller so that its temperature increases to 100°C. In a specific embodiment, the heating element may be controlled by a controller so that its temperature increases to 105°C.

[0269] In step (iv), incubation may be performed for a sufficient amount of time to extract nucleic acids. The incubation time may vary depending on various factors, such as the type of organism from which nucleic acids are to be extracted, e.g., the type of pathogen, the type of biological sample, and the components of the cell lysis composition. A person skilled in the art may determine an appropriate incubation time based on the correlation between the incubation time and the concentration of the extracted nucleic acids, or the correlation between the incubation time and the efficiency or sensitivity of the nucleic acid amplification reaction.

[0270] In one embodiment, the incubation of step (iv) is performed for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, or for a time in between. In a specific embodiment, the incubation of step (iv) is performed for about 10 minutes.

[0271] The method of the present disclosure does not include the extraction of nucleic acids other than the lysis of cells using the aforementioned cell lysis composition. Specifically, the method of the present disclosure does not include steps for purifying nucleic acids, such as binding nucleic acids to a solid support, washing, or eluting, after cell lysis.

[0272] The method of the present disclosure produces a cell lysate, which can be directly applied to a nucleic acid amplification reaction.

[0273] The present invention will be described in more detail below through examples. These examples are intended to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention as set forth in the appended claims is not limited by these examples.

[0274] Examples

[0275] Example 1: Determination of Optimal Concentrations of Cell Lysation Components

[0276] To determine the optimal concentrations of each of the cell lysis components—(i) guanidine hydrochloride (GuHCl), (ii) cetyltrimethylammonium bromide (CTAB), (iii) polyethylene glycol 200 (PEG200), (iv) n-lauroylsarcosine (NLS), and (v) polyoxyethylene sorbitan monolaurate (Tween 20)—various concentrations of each component were added to nucleic acid amplification reagents to investigate their effects on PCR performance (Ct value, and RFU (relative fluorescence unit)).

[0277] First, Streptococcus piogenes, a group A Streptococcus (GAS) obtained from ZeptoMetrix, into an ESwab® 480C (Copan Diagnostics Inc) tube (filled with 1 mL of liquid Amies transport medium) Streptococcus pyogenes ) 6.02 X 10 6Spiking was performed at a concentration of CFU / mL.

[0278] Subsequently, various concentrations of the following five components were added as experimental groups: (i) for GuHCl, 30 mM, 100 mM, 300 mM, 500 mM, and 1000 mM; (ii) for CTAB, 0.1 wt%, 0.3 wt%, 0.5 wt%, 1.0 wt%, and 1.5 wt%; (iii) for PEG200, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, and 70 wt%; (iv) for NLS, 0.05 wt%, 0.1 wt%, 0.3 wt%, 0.5 wt%, 1 wt%, and 1.5 wt%; and (v) for Tween 20, 5 wt%, 10 wt%, 15 wt%, 20 wt%, and 25 wt%.

[0279] Meanwhile, for comparison, TE buffer (Tris 10 mM and EDTA 1 mM, pH 8.0) was added as a control instead of the above components.

[0280] Afterwards, the above experimental group and control group were each incubated at 25°C for 10 minutes to lyse the cells of S. piogenes.

[0281] Subsequently, a nucleic acid amplification reagent was prepared consisting of primers and probes for amplifying / detecting the target nucleic acid of the above-mentioned S. piogenes and a PCR enzyme mix (including Hot-start Taq polymerase and dNTPs). The primers and probes were designed to mimic slightly modified sequences under the CDC (Centers for Disease Control and Prevention) guidelines (Streptococcus Laboratory Resources and Protocols) and are shown in Table 1 below.

[0282] designation order Sequence number spy-F 5'-GCACTCGCTACTATTTCTTACCTCAA-3' 1 spy-R 5'-GTCACAATGTCTTGGAAACCAGTAAT-3' 2 spy-P 5'-FAM-CCGCAACTCATCAAGGATTTCTGTTACCA-BHQ1-3' 3

[0283] Subsequently, a reaction mixture was prepared by mixing 5 μL of each of the experimental group or control group with 15 μL of the nucleic acid amplification reagent. The tube containing the reaction mixture was placed in a CFX96 real-time PCR thermal cycler (Bio-Rad) to amplify the target nucleic acid by performing 5 cycles of 5 seconds at 95°C and 20 seconds at 60°C, and 40 cycles of 2 seconds at 95°C and 5 seconds at 60°C. The signal was measured at 60°C for each cycle to obtain an amplification curve. Subsequently, a threshold value of 110 RFU (Relative Fluorescence Unit) was applied to the amplification curve to calculate the Ct value, and the RFU at the last cycle was recorded.

[0284] <1-1> GuHCl

[0285] The results of PCR reactions using different concentrations of GuHCl are shown in Table 2 below. Each result is the average of three replicates.

[0286] reaction control group GuHCl 30 mM 100 mM 300 mM 500 mM 1000 mM Ct value #1 33.59 33.36 33.65 37.31 N / A N / A #2 32.85 33.04 33.49 37.14 N / A N / A #3 32.69 32.89 33.48 37.52 N / A N / A average 33.04 33.10 33.54 37.32 - - Experimental group vs. Control group - 0.05 0.50 4.28 - - Standard deviation 0.48 0.24 0.10 0.19 - - RFU #1 1306.27 1372.05 1459.66 997.49 11.92 3.88 #2 1496.99 1527.31 1451.13 1198.61 9.54 1.53 #3 1395.81 1487.47 1434.55 1034.57 10.15 7.17 average 1399.69 1462.28 1448.45 1076.89 10.54 4.19 Experimental group vs. Control group - 62.59 48.76 -322.80 -1389.15 -1395.50

[0287] As shown in Table 2 above, when GuHCl was added in an amount of 500 mM or more, no Ct value was produced and the RFU value was found to be significantly lower, which demonstrates the inhibitory effect of GuHCl of 500 mM or more on the PCR reaction.

[0288] The above results show that it is desirable to add GuHCl in an amount of about 30 to about 300 mM.

[0289] <1-2> CTAB

[0290] The results of PCR reactions using different concentrations of CTAB are shown in Table 3 below. Each result is the average of three replicates.

[0291] reaction control group CTAB 0.05 wt% 0.1 wt% 0.5 wt% 1.0 wt% 1.5 wt% Ct value #1 31.72 27.25 28.42 N / A N / A N / A #2 31.40 27.34 28.44 N / A N / A N / A #3 31.44 27.25 27.38 N / A N / A N / A average 31.52 27.28 28.08 - - - Experimental group vs. Control group -　 -4.24 -3.44 - - - Standard deviation 0.17 0.05 0.61 - - - RFU #1 2335 2900 2612 14 -9 -15 #2 2427 2906 2648 22 -3 -8 #3 2583 2820 2780 18 -3 -7 average 2448 2875 2680 18 -5 -10 Experimental group vs. Control group - 427.00 231.67 -2430.20 -2453.15 -2458.25

[0292] As shown in Table 3 above, when 0.5 wt%, 1.0 wt%, and 1.5 wt% of CTAB were added, it was found that no Ct value was produced and the RFU value was significantly lowered, which demonstrates the inhibitory effect of CTAB of 0.5 wt% or more on the PCR reaction.

[0293] The above results show that it is preferable to add CTAB in an amount of about 0.05 weight% to less than 0.5 weight%.

[0294] <1-3> PEG200

[0295] The results of PCR reactions using different concentrations of PEG200 are shown in Tables 4 and 5 below. Each result is the average of three replicates.

[0296] reaction control group PEG200 5 wt% 10 wt% 15 wt% 20 wt% 25 wt% Ct value #1 33.35 32.92 32.29 32.60 31.84 32.59 #2 32.27 32.56 32.14 32.12 32.25 32.37 #3 32.54 32.86 32.30 32.25 32.32 32.49 average 32.72 32.78 32.24 32.32 32.14 32.48 Experimental group vs. Control group -　 0.06 -0.48 -0.40 -0.58 -0.24 Standard deviation 0.56 0.19 0.09 0.25 0.26 0.11 RFU #1 1180.20 1307.15 1423.39 1264.36 1318.41 1241.85 #2 1192.16 1413.18 1385.73 1419.79 1270.66 1393.67 #3 1199.84 1309.07 1375.36 1320.24 1266.19 1359.10 average 1190.73 1343.13 1394.82 1334.80 1285.09 1331.54 Experimental group vs. Control group - 152.40 204.09 144.06 94.36 140.81

[0297] reaction control group PEG200 30 wt% 40 wt% 50 wt% 60 wt% 70 wt% 80 wt% Ct value #1 31.72 30.84 30.90 31.16 29.24 29.56 29.18 #2 31.40 30.77 31.43 30.75 29.53 30.09 29.95 #3 31.44 30.85 31.49 30.74 29.99 28.88 28.76 average 31.52 30.82 31.27 30.88 29.58 29.51 29.30 Experimental group vs. Control group -　 -0.70 -0.25 -0.64 -1.94 -2.01 -2.22 Standard deviation 0.17 0.04 0.33 0.24 0.38 0.61 0.43 RFU #1 2335 2335 2384 2097 2328 2137 2103 #2 2427 2457 2324 2308 2301 2041 2067 #3 2583 2359 2226 2241 2303 2377 2331 average 2448 2384 2311 2215 2311 2185 2167 Experimental group vs. Control group - -64.67 -137.00 -233.00 -137.67 -263.33 -280.62

[0298] As shown in Tables 4 and 5 above, when PEG200 was added, the Ct value decreased, but the RFU value decreased slightly. However, it was found that PEG200 did not have a significant effect on the PCR reaction despite the addition of a large amount.

[0299] The above results show that 5% to 80% by weight of PEG200 does not significantly affect the PCR reaction.

[0300] <1-4> NLS

[0301] The results of PCR reactions using different concentrations of NLS are shown in Table 6 below. Each result is the average of three replicates.

[0302] reaction control group NLS 0.05 wt% 0.1 wt% 0.3 wt% 0.5 wt% 1 wt% 1.5 wt% Ct value #1 33.35 32.86 33.45 32.91 34.33 N / A N / A #2 32.27 31.96 33.24 32.74 34.21 N / A N / A #3 32.54 32.14 32.95 33.01 34.00 N / A N / A average 32.72 32.32 33.21 32.89 34.18 - - Experimental group vs. Control group -　 -0.40 0.49 0.17 1.46 - - Standard deviation 0.56 0.17 0.25 0.14 0.17 - - RFU #1 1180.20 1386.49 1321.44 1428.90 1342.39 1.57 1.58 #2 1192.16 1441.69 1447.40 1390.89 1410.09 1.97 1.41 #3 1199.84 1366.72 1379.19 1371.27 1388.29 5.14 2.51 average 1190.73 1405.31 1382.67 1397.02 1380.25 2.89 1.83 Experimental group vs. Control group 209.32 152.40 191.94 206.29 189.52 -1187.84 -1188.90

[0303] As shown in Table 6 above, the addition of 1 wt% and 1.5 wt% of NLS did not yield Ct values ​​and showed a significant decrease in RFU values, which demonstrates the inhibitory effect of 1 wt% to 1.5 wt% of NLS on the PCR reaction.

[0304] The above results show that it is preferable to add NLS in an amount of about 0.05 weight% to less than 1 weight%.

[0305] <1-5> Tween 20

[0306] The results of PCR reactions using different concentrations of Tween 20 are shown in Table 7 below. Each result is the average of three replicates.

[0307] reaction control group Tween 20 5 wt% 10 wt% 15 wt% 20 wt% 25 wt% Ct value #1 33.35 31.19 31.44 31.45 31.41 31.10 #2 32.27 31.19 31.16 30.83 31.28 30.78 #3 32.54 31.75 31.45 31.12 31.27 31.15 average 32.72 31.38 31.35 31.13 31.32 31.01 Experimental group vs. Control group - -1.34 -1.37 -1.59 -1.40 -1.71 Standard deviation 0.56 0.32 0.16 0.31 0.08 0.20 RFU #1 1180.20 1156.60 1045.55 1074.70 912.31 914.67 #2 1192.16 1131.86 1146.89 1032.42 998.57 949.03 #3 1199.84 1175.17 1063.07 983.14 947.20 909.12 average 1190.73 1154.54 1085.17 1030.09 952.69 924.27 Experimental group vs. Control group 0.00 -36.19 -105.56 -160.65 -238.04 -266.46

[0308] As shown in Table 7 above, when Tween 20 was added in an amount of 20 wt% or 25 wt%, the RFU value was slightly lowered.

[0309] The above results show that it is preferable to add Tween 20 in an amount of about 5% to 15% by weight.

[0310] Example 2: Effect of pH of cell lysis composition

[0311] In order to examine the effect of pH on the cell lysis composition according to the present disclosure, cell lysis compositions having the same components but different pH were prepared.

[0312] First, 6.02 x 10⁶ Streptococcus piogenes were placed in the tube of the ESwab® 480C (Copan Diagnostics Inc). 6 Spiked at a concentration of CFU / mL.

[0313] Subsequently, as an experimental group, 10 mM Tris, 1 mM EDTA, 100 mM GuHCl, 0.05 wt% NLS, 5 wt% Tween 20, 15 wt% PEG8000, and 5 wt% EtOH were mixed, and NaOH was added to adjust the pH of the mixture to 8, 9, 10, 11, and 12, respectively. Then, 100 μL of the mixture was added to each tube and incubated at 25°C for 10 minutes to lyse the cells of S. piogenes.

[0314] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0315] Afterwards, the above experimental group or control group was subjected to PCR using a nucleic acid amplification reagent as in Example 1 to obtain an amplification curve, and the Ct value was calculated from the amplification curve.

[0316] The above results are shown in Table 8 below. Each result is the average of three repetitions.

[0317] control group Experimental group pH 8 Experimental group pH 9 Experimental group pH 10 Experimental group pH 11 Experimental group pH 12 #1 31.51 31.36 31.05 30.77 32.99 31.17 #2 31.38 31.30 31.45 30.52 32.18 32.09 #3 31.06 31.19 31.94 32.00 31.61 31.73 average 31.32 31.28 31.48 31.09 32.26 31.66 Experimental group vs. Control group - -0.03 0.16 -0.22 0.94 0.35 Standard deviation 0.23 0.09 0.44 0.79 0.70 0.46

[0318] As shown in Table 8 above, the pH of the cell lysis composition was found to have little effect on the PCR reaction. Given that the pH of the most widely used Tris buffer is 8 to 9, it was determined that it would be desirable to adjust the pH of the composition to 8.

[0319] Example 3: Effects of the CTAB and PEG combination

[0320] To examine the effect of the combination of CTAB and PEG on cell lysis, a composition containing the combination of CTAB and PEG was compared with other compositions.

[0321] First, 6.02 x 10⁶ Streptococcus piogenes were placed in the tube of the ESwab® 480C (Copan Diagnostics Inc). 6 Spiked at a concentration of CFU / mL.

[0322] Subsequently, as experimental groups, (i) a composition containing GuHCl and PEG (Experimental Group 1), (ii) a composition containing low concentrations of CTAB and PEG (Experimental Group 2), (iii) a composition containing high concentrations of CTAB and PEG (Experimental Group 3), and (iv) a composition containing only PEG without GuHCl and CTAB (Experimental Group 4) were prepared. Then, 100 μL of each composition was added to a tube and incubated at 25°C for 10 minutes to lyse S. piogenes cells.

[0323] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0324] Afterwards, the above experimental group or control group was subjected to PCR using a nucleic acid amplification reagent as in Example 1 to obtain an amplification curve, and the Ct value was calculated from the amplification curve.

[0325] The above results are shown in Table 9 below. Each result is the average of three repetitions.

[0326] reaction control group Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Reagent composition Tris 10 mM 10 mM 10 mM 10 mM EDTA 1 mM 1 mM 1 mM 1 mM GuHCl 100 mM - - - CTAB - 0.1 wt% 0.5 wt% - NLS 0.05 wt% 0.05 wt% 0.05 wt% - Tween 20 5 wt% 5 wt% 5 wt% 5 wt% PEG200 60 wt% - - 60 wt% PEG8000 - 15 wt% 15 wt% - pH 8 8 8 8 #1 31.44 30.26 28.40 28.69 31.19 #2 31.20 29.92 28.55 28.45 31.49 #3 31.01 29.75 28.44 28.72 32.11 average 31.22 29.98 28.46 28.62 31.60 Experimental group vs. Control group - -1.24 -2.76 -2.60 0.38 Standard deviation 0.22 0.26 0.08 0.15 0.47

[0327] As shown in Table 9 above, experimental groups 2 and 3 containing CTAB and PEG showed lower Ct values ​​compared to experimental group 1 containing GuHCl and PEG, experimental group 4 containing only PEG, and the control group, which demonstrates the superior PCR reaction of experimental groups 2 and 3 containing CTAB and PEG.

[0328] The above results show that using a cell lysis composition containing a combination of CTAB and PEG can improve the detection performance of target nucleic acids.

[0329] Example 4: Effects of GuHCl

[0330] To examine the effect of GuHCl on cell lysis, a composition containing GuHCl was compared with a composition not containing GuHCl.

[0331] First, 6.02 x 10⁶ Streptococcus piogenes were placed in the tube of the ESwab® 480C (Copan Diagnostics Inc). 6 Spiked at a concentration of CFU / mL.

[0332] Subsequently, as experimental groups, (i) a composition containing low concentrations of CTAB and PEG without GuHCl (Experimental Group 1), (ii) a composition containing high concentrations of CTAB and PEG without GuHCl (Experimental Group 2), (iii) a composition containing low concentrations of GuHCl, low concentrations of CTAB and PEG (Experimental Group 3), and (iv) a composition containing high concentrations of GuHCl, low concentrations of CTAB and PEG (Experimental Group 4) were prepared. Then, 100 μL of each composition was added to a tube and incubated at 25°C for 10 minutes to lyse S. piogenes cells.

[0333] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0334] Afterwards, the above experimental group and control group were subjected to PCR using nucleic acid amplification reagents as in Example 1 to obtain amplification curves, and the Ct value and RFU at the last cycle were recorded.

[0335] The above results are shown in Table 10 below. Each result is the average of three repetitions.

[0336] reaction control group Experimental Group 1 Experimental Group 2 Experimental group 3 Experimental group 4 Reagent composition Tris 10 mM 10 mM 10 mM 10 mM EDTA 1 mM 1 mM 1 mM 1 mM GuHCl - - 100 mM 250 mM CTAB 0.1 wt% 0.5 wt% 0.1 wt% 0.1 wt% NLS 0.05 wt% 0.05 wt% 0.05 wt% 0.05 wt% Tween 20 5 wt% 5 wt% 5 wt% 5 wt% PEG8000 15 wt% 15 wt% 15 wt% 15 wt% pH 8 8 8 8 Ct value #1 30.50 28.88 29.06 29.19 30.04 #2 31.20 29.03 28.49 29.02 29.87 #3 31.29 28.68 28.43 29.02 30.08 average 31.00 28.87 28.66 29.08 30.00 Experimental group vs. Control group - -2.13 -2.34 -1.92 -1.00 Standard deviation 0.43 0.18 0.34 0.10 0.11 RFU #1 1340.14 1066.46 869.79 1154.41 1258.12 #2 1343.51 1069.89 927.94 1166.18 1212.80 #3 1328.77 1073.35 877.35 1153.35 1191.89 average 1337.48 1069.90 891.70 1157.98 1220.94 Experimental group vs. Control group - -267.57 -445.78 -179.50 -116.54 Standard deviation 1340.14 1066.46 869.79 1154.41 1258.12

[0337] As shown in Table 10 above, experimental groups 1 and 2, which contain CTAB and PEG but do not contain GuHCl, showed a decrease in Ct value but a decrease in RFU value. In contrast, experimental groups 3 and 4, which contain all of GuHCl, CTAB, and PEG, showed an improvement in RFU value due to the addition of GuHCl compared to experimental groups 1 and 2.

[0338] The above results show that the decrease in RFU values ​​caused by the use of CTAB and PEG can be improved by the addition of GuHCl.

[0339] Example 5: Effects of PEG200

[0340] To examine the effect of the molecular weight of PEG on cell lysis, compositions containing PEG200 or PEG8000 were compared.

[0341] First, 6.02 x 10⁶ Streptococcus piogenes were placed in the tube of the ESwab® 480C (Copan Diagnostics Inc). 6 Spiked at a concentration of CFU / mL.

[0342] Subsequently, as experimental groups, (i) a composition containing PEG8000 (experimental group 1), and (ii) a composition containing PEG200 (experimental group 2) were prepared.

[0343] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0344] Afterwards, the above experimental group and control group were subjected to PCR using nucleic acid amplification reagents as in Example 1 to obtain amplification curves, and the Ct value and RFU at the last cycle were recorded.

[0345] The above results are shown in Table 11 below. Each result is the average of three repetitions.

[0346] reaction control group Experimental Group 1 Experimental Group 2 Reagent composition Tris 10 mM 10 mM EDTA 1 mM 1 mM GuHCl 100 mM 100 mM CTAB 0.1 wt% 0.1 wt% NLS 0.05 wt% 0.05 wt% Tween 20 5 wt% 5 wt% PEG200 - 60 wt% PEG8000 15 wt% - pH 8 8 Ct value #1 31.37 29.58 29.38 #2 31.02 29.25 29.58 #3 30.72 29.05 29.55 average 31.04 29.29 28.50 Experimental group vs. Control group - -1.74 -2.53 Standard deviation 0.33 0.27 0.12 RFU #1 1268.87 1094.75 1013.93 #2 1325.63 1092.26 1030.55 #3 1334.75 1112.63 983.36 average 1309.75 1099.88 1009.28 Experimental group vs. Control group - -209.87 -300.47

[0347] As shown in Table 11 above, experimental group 2, containing PEG200 at a concentration of 60 wt%, was found to have superior amplification performance of the target nucleic acid compared to experimental group 1, containing PEG8000 at a concentration of 15 wt% (Ct value decreased).

[0348] In addition, the solution of experimental group 1 containing PEG8000 (powder state) at a concentration of 15 wt% showed poor handling ability due to many bubbles and high viscosity, whereas PEG200 (liquid state) showed good handling ability.

[0349] Example 6: Effects of Tris and PEG200

[0350] To examine the effects of Tris and PEG200 on cell lysis, compositions containing different concentrations of Tris and PEG200 were compared.

[0351] First, 6.02 x 10⁶ Streptococcus piogenes were placed in the tube of the ESwab® 480C (Copan Diagnostics Inc). 6 Spiked at a concentration of CFU / mL.

[0352] Subsequently, as experimental groups, (i) a composition containing 10 mM Tris and 60 wt% PEG200 (experimental group 1), (ii) a composition containing 50 mM Tris and 60 wt% PEG200 (experimental group 2), and (iii) a composition containing 10 mM Tris (experimental group 3) were prepared.

[0353] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0354] Afterwards, the above experimental group and control group were subjected to PCR using nucleic acid amplification reagents as in Example 1 to obtain amplification curves, and the Ct value and RFU at the last cycle were recorded.

[0355] The above results are shown in Table 12 below. Each result is the average of three repetitions.

[0356] reaction control group Experimental Group 1 Experimental Group 2 Experimental Group 2 Reagent composition Tris 10 50 10 EDTA 1 1 1 GuHCl 100 100 100 CTAB 0.1 0.1 0.1 NLS 0.05 0.05 0.05 Tween 20 5 5 5 PEG200 60 60 - pH 8 8 8 Ct value #1 33.02 30.76 31.10 32.96 #2 32.88 30.76 31.26 32.94 #3 32.88 30.72 30.70 32.90 average 32.93 30.75 31.02 32.93 Experimental group vs. Control group - -2.14 -1.86 0.05 Standard deviation 0.08 0.02 0.29 0.03 RFU #1 2181.17 1887.89 1999.24 1607.10 #2 2260.74 1882.38 2181.53 1847.46 #3 2262.68 1919.09 2068.28 1538.23 average 2234.86 1896.45 2083.02 1664.26 Experimental group vs. Control group - -366.23 -179.66 -598.42

[0357] As shown in Table 12 above, experimental group 1, containing Tris at a concentration of 10 mM, showed a Ct value approximately 0.3 lower than experimental group 2, containing Tris at a concentration of 50 mM. This indicates that using a small amount of Tris at 10 mM is advantageous for the detection of target nucleic acids.

[0358] Meanwhile, experimental groups 1 and 2 containing PEG200 were found to have lower Ct values ​​and higher RFU values ​​compared to experimental group 3 not containing PEG200. The above results show that the addition of PEG200 is advantageous for cell lysis.

[0359] Example 7: Confirmation of Analytical Sensitivity of Cell Lysis Composition

[0360] To confirm the analytical sensitivity of PCR using the cell lysis composition according to the present disclosure, three strains of causative pathogens of pharyngitis, namely Streptococcus piogenes, Streptococcus disgalactiae subspecies equisimilis ( Streptococcus dysgalactiae subsp. Equisimilis ), and Arcanobacterium hamolitiscum ( Arcanobacterium haemolyticum After obtaining the target nucleic acid using the cell lysis composition from the above, the target nucleic acid was immediately subjected to a PCR reaction.

[0361] Specifically, a 5-fold serial dilution of Streptococcus piogenes obtained from ZeptoMetrix, namely 6.02 x 10⁻⁶ 6 CFU / mL to 6.02 X 10⁻⁶ 2 CFU / mL, 5-fold serial dilution of Streptococcus disgalactiae subspecies equityimilis, i.e., 4.14 x 10⁻⁶ 4 CFU / mL to 4.14 X 10⁻⁶ 0 CFU / mL, and a 5-fold serial dilution of Arcanobacterium hamolitiscum, i.e., 6.12 x 10⁻⁶ 4 CFU / mL to 6.12 X 10⁻⁶ 1 Each CFU / mL was spiked into the first to fifth tubes of ESwab® 480C (Copan Diagnostics Inc).

[0362] Subsequently, 100 μL of the above sample and 100 μL of a cell lysis composition according to the present disclosure (Tris 10 mM, EDTA 1 mM, GuHCl 100 mM, CTAB 0.1 wt%, NLS 0.05 wt%, Tween 20 5 wt% and PEG200 60 wt%; pH 8) as an experimental group were added, and then the cells of each strain were lysed by incubating at 25°C for 10 minutes.

[0363] Meanwhile, as a control group for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract the target nucleic acid.

[0364] Subsequently, a nucleic acid amplification reagent was prepared consisting of primers and probes for amplifying / detecting the target nucleic acids of each of the above strains and a PCR enzyme mix (including Hot-start Taq polymerase and dNTPs). The specific sequences of the primers and probes are shown in Table 13 below.

[0365] designation order Sequence number spy-F 5'-GCACTCGCTACTATTTCTTACCTCAA-3' 1 spy-R 5'-GTCACAATGTCTTGGAAACCAGTAAT-3' 2 spy-P 5'-FAM-CCGCAACTCATCAAGGATTTCTGTTACCA-BHQ1-3' 3 SDSE-F 5'-CGCGAATCATTGCTCTGTGG-3' 4 SDSE-R 5'-AACCGATGGTGTGGAAAGCA-3' 5 SDSE-P 5'-HEX-ACCGCAACCATTTCTCGATTTGCT-BHQ1-3' 6 AH-F 5'-CGCGAACGCTCTGGAAATT-3' 7 AH-R 5'-GCCGTATCGCCAGCACTAGT-3' 8 AH-P 5'-C610-ATTTTACTGCGTGGCGCC-BHQ1-3' 9

[0366] Subsequently, a reaction mixture was prepared by mixing 5 μL of the experimental or control group with 15 μL of the nucleic acid amplification reagent. The tube containing the reaction mixture was placed in a CFX96 real-time PCR thermal cycler (Bio-Rad) and the target nucleic acid was amplified by applying 5 cycles of 5 seconds at 95°C and 20 seconds at 60°C, and 40 cycles of 2 seconds at 95°C and 5 seconds at 60°C. The signal was measured at 60°C for each cycle to obtain an amplification curve. Subsequently, a threshold value of 110 RFU (Relative Fluorescence Unit) was applied to the amplification curve to calculate the Ct value.

[0367] The above results are shown in Table 14 below. Each result is the average of three repetitions.

[0368] strain density control group experimental group Ct value average Ct value average Streptococcus pionogenes 6.02 X 10 6 CFU / mL 29.50 29.50 27.68 27.79 29.54 27.51 29.47 28.17 6.02 X 10 5 CFU / mL 32.72 32.94 31.19 31.28 32.83 30.94 33.28 31.72 6.02 X 10 4 CFU / mL 35.87 35.86 35.06 34.94 34.99 34.38 36.73 35.39 6.02 X 10 3 CFU / mL N / A - 37.97 38.95 N / A 41.34 N / A 37.53 6.02 X 10 2 CFU / mL N / A - 44.62 - N / A N / A N / A N / A

[0369] strain density control group experimental group Ct value average Ct value average Streptococcus disgalactiae subspecies equityimilis 4.14 X 10 4 CFU / mL 31.93 31.95 26.95 27.67 31.99 28.01 31.94 28.06 4.14 X 10 3 CFU / mL 35.74 36.01 30.77 30.85 37.11 31.09 35.16 30.69 4.14 X 10 2 CFU / mL N / A - 34.06 34.21 N / A 34.39 N / A 34.18 4.14 X 10 1 CFU / mL N / A - 38.78 - N / A 35.36 N / A N / A 4.14 X 10 0 CFU / mL N / A - N / A - N / A 37.03 N / A N / A

[0370] strain density control group experimental group Ct value average Ct value average Arcanobacterium hamolitiscum 6.12 X 10 5 CFU / mL 26.98 26.90 26.17 25.98 26.90 25.81 26.82 25.96 6.12 X 10 4 CFU / mL 30.79 30.66 29.30 29.37 30.75 29.50 30.44 29.30 6.12 X 10 3 CFU / mL 34.64 33.99 32.96 34.47 33.70 34.94 33.63 35.52 6.12 X 10 2 CFU / mL 37.15 - N / A - 36.05 35.98 N / A 34.22 6.12 X 10 1 CFU / mL N / A - N / A - N / A N / A N / A N / A

[0371] As shown in Table 14 above, the control group is 6.02 X 10 6 CFU / mL to 6.02 X 10⁻⁶ 4 While it can detect Streptococcus piogenes at a concentration of CFU / mL, the experimental group was 6.02 X 10 6 CFU / mL to 6.02 X 10⁻⁶ 3 Streptococcus piogenes at a concentration of CFU / mL could be detected. The above results demonstrate that a PCR reaction using a cell lysis composition according to the present disclosure has higher analytical sensitivity in detecting Streptococcus piogenes compared to a PCR reaction using a conventional extraction method.

[0372] As shown in Table 15 above, the control group is 4.14 X 10 4 CFU / mL to 4.14 X 10⁻⁶ 3While it can detect Streptococcus disgalactiae subspecies equityimilis at a concentration of CFU / mL, the experimental group was 4.14 X 10 4 CFU / mL to 4.14 X 10⁻⁶ 2 Streptococcus disgalactiae subspecies equisimyls could be detected at a concentration of CFU / mL. The above results demonstrate that a PCR reaction using a cell lysis composition according to the present disclosure has higher analytical sensitivity in detecting Streptococcus disgalactiae subspecies equisimyls compared to a PCR reaction using a conventional extraction method.

[0373] As shown in Table 16 above, the control group is 6.12 x 10 5 CFU / mL to 6.12 X 10⁻⁶ 3 It can detect Arcanobacterium hamolitiscum at a concentration of CFU / mL, and the experimental group is also 6.12 X 10 5 CFU / mL to 6.12 X 10⁻⁶ 3 It was possible to detect Arcanobacterium hamolitisum at a concentration of CFU / mL. The above results demonstrate that a PCR reaction using the cell lysis composition according to the present disclosure has analytical sensitivity equivalent to that of conventional extraction methods in detecting Arcanobacterium hamolitisum.

[0374] In summary, a PCR reaction using the cell lysis composition according to the present disclosure has superior or equivalent analytical sensitivity compared to a PCR reaction using conventional nucleic acid extraction, which demonstrates that the cell lysis composition according to the present disclosure can enable a simple and rapid PCR reaction without the need for nucleic acid purification.

[0375] Example 8: Effect of pre-incubation of a cell lysis composition in an automated liquid handling system

[0376] In order to examine the effect of the nucleic acid extraction method according to the present disclosure, which includes pre-incubation of a cell lysis composition in an automated liquid handling system, nucleic acids were extracted from three strains of causative pathogens of pharyngitis, namely Streptococcus piogenes, Streptococcus disgalactiae subspecies equisimilis, and Arcanobacterium hamolitiscum by the method of one embodiment of the present disclosure, and then immediately subjected to a PCR reaction.

[0377] Specifically, each tube containing a cell lysis composition and each tube containing a biological sample were mounted on an automated liquid handling system (Seegene STARlet, dimensions: 1124 (W) x 795 (D) x 903 (H) mm).

[0378] The cell lysis composition above consists of Tris 10 mM, EDTA 1 mM, GuHCl 100 mM, CTAB 0.1 wt%, NLS 0.05 wt%, Tween 20 5 wt% and PEG200 60 wt%; pH 8.

[0379] Streptococcus piogenes 6.02 X 10⁻⁶ obtained from ZeptoMetrix 6 CFU / mL, Streptococcus disgalactiae subspecies equityimilis 4.14 X 10⁶ 4 CFU / mL, and Arcanobacterium hamolitiscum 6.12 X 10 5 After spiking each CFU / mL into the first to third tubes of the ESwab® 480C (Copan Diagnostics Inc), some of the spiked solution was transferred to a new tube to prepare a biological sample.

[0380] Subsequently, an empty 96 deep-well plate was mounted on a Hamilton heater shaker within an automated liquid handling system.

[0381] Subsequently, the temperature of the Hamilton heater shaker was increased to a predetermined temperature of 100°C by the controller of the automated liquid handling system to heat the empty 96 deep well plate.

[0382] Next, 50 μL of the cell lysis composition was dispensed into each well of the empty 96-deep-well plate by the controller of the automated liquid handling system.

[0383] Next, the temperature of the Hamilton heater shaker was increased to a predetermined temperature of 100°C by the controller of the automated liquid handling system, and the cell lysis composition was heated for 10 minutes. During the heating, the maximum temperature of the cell lysis composition was measured. It was confirmed that the cell lysis composition was maintained at approximately 58-60°C.

[0384] Next, 50 μL of the biological sample was dispensed into each well of a 96-deep-well plate containing the cell lysis composition by a controller of an automated liquid handling system. The biological sample and the cell lysis composition were thoroughly mixed by pipetting using a pipetting channel.

[0385] Next, the temperature of the Hamilton heater shaker was increased to a predetermined temperature of 100°C by the controller of the automated liquid handling system and the mixture was heated for 10 minutes, and as a result, a cell lysate was obtained.

[0386] Subsequently, a nucleic acid amplification reagent was prepared consisting of primers and probes for amplifying / detecting the target nucleic acid of each of the above strains and a PCR enzyme mix (including Hot-start Taq polymerase and dNTPs). The sequences of the primers and probes are as described in Table 13 of Example 7.

[0387] Subsequently, a reaction mixture was prepared by mixing 5 μL of the cell lysate and 15 μL of the nucleic acid amplification reagent. The tube containing the reaction mixture was placed in a CFX96 real-time PCR thermal cycler (Bio-Rad) and the target nucleic acid was amplified by applying 5 cycles of 5 seconds at 95°C and 20 seconds at 60°C, and 40 cycles of 2 seconds at 95°C and 5 seconds at 60°C. The signal was measured at 60°C for each cycle to obtain an amplification curve. Subsequently, a threshold value of 110 RFU (Relative Fluorescence Unit) was applied to the amplification curve to calculate the Ct value.

[0388] The above results are shown in Table 17 below. Each result is the average of three repetitions.

[0389] strain Ct value Streptococcus pionogenes 27.87 Streptococcus disgalactiae subspecies equityimilis 29.69 Arcanobacterium hamolitiscum 26.10

[0390] As shown in Table 17 above, the cell lysate obtained by the method of the present disclosure, which includes pre-incubation of the cell lysate composition, was found to provide excellent detection results without inhibition of the nucleic acid amplification reaction.

[0391] Comparative Example 9: Effect of pre-incubation of biological samples in an automated liquid handling system

[0392] To compare the effect of pre-incubation of the cell lysis composition with that of the biological sample, the biological sample was pre-incubated (the cell lysis composition was not pre-incubated), nucleic acids were extracted, and then applied to a nucleic acid amplification reaction.

[0393] Specifically, the empty 96 deep-well plate was heated by increasing the temperature of the Hamilton heater shaker to a predetermined temperature of 100°C by the controller of the same automated liquid handling system used in Example 8.

[0394] Next, 50 μL of the biological sample was dispensed into each well of the empty 96-deep-well plate by the controller of the automated liquid handling system.

[0395] Next, the temperature of the Hamilton heater shaker was increased to a predetermined temperature of 100°C by the controller of the automated liquid handling system, and the biological sample was heated for 10 minutes. During the heating, the maximum temperature of the biological sample was measured. It was confirmed that the biological sample was maintained at approximately 58-60°C.

[0396] Next, 50 μL of cell lysis composition was dispensed into each well of a 96-deep-well plate containing the biological sample by a controller of an automated liquid handling system. The biological sample and the cell lysis composition were thoroughly mixed by pipetting using a pipetting channel.

[0397] Next, the temperature of the Hamilton heater shaker was increased to a predetermined temperature of 100°C by the controller of the automated liquid handling system and the mixture was heated for 10 minutes, and as a result, a cell lysate was obtained.

[0398] Afterward, real-time PCR was performed in the same manner as in Example 8, and the Ct value was calculated.

[0399] The above results are shown in Table 18 below. Each result is the average of three repetitions.

[0400] strain Ct value Example 8 - Comparative Example 9 Streptococcus pionogenes 29.29 - 1.42 Streptococcus disgalactiae subspecies equityimilis 32.00 - 2.31 Arcanobacterium hamolitiscum 27.64 - 1.54

[0401] As shown in Table 18 above, it was confirmed that the nucleic acid extraction method including the step of pre-incubating a biological sample (Comparative Example 9) exhibited a higher Ct value compared to the nucleic acid extraction method including the step of pre-incubating a cell lysis composition (Example 8). This demonstrates that pre-incubating a biological sample results in lower efficiency of the nucleic acid amplification reaction compared to pre-incubating a cell lysis composition.

[0402] Example 10: Effect of pre-incubation time of a cell lysis composition in an automated liquid handling system

[0403] To investigate the effect of pre-incubation time of a cell lysis composition on a nucleic acid amplification reaction in an automated liquid handling system, the cell lysis composition was pre-incubated for different times, and the obtained nucleic acid was applied to a real-time PCR reaction as in Example 8.

[0404] Specifically, the process described in Example 8 was repeated, except that three strains were spiked in the following amounts:

[0405] - Streptococcus piogenes (ZeptoMetrix): 6.02 X 10⁻⁶ 5 CFU / mL, 6.02 X 10 4 CFU / mL, and 6.02 X 10⁻⁶ 3 CFU / mL

[0406] - Streptococcus disgalactiae subspecies equityimilis (ATCC): 1.4 X 10 5 CFU / mL, 1.4 X 10 4 CFU / mL, and 1.4 X 10 3 CFU / mL

[0407] - Arcanobacterium hamolitiscum (ATCC): 1 X 10 4 CFU / mL, 1 X 10 3 CFU / mL, and 1 X 10 2 CFU / mL

[0408] The results for Streptococcus piogenes, Streptococcus disgalactiae subspecies equityimilis, and Arcanobacterium haemolitiscum are shown in Tables 19, 20, and 21, respectively. Each result is the mean of three replicates.

[0409] density Experimental group 1 (preheating 5 minutes) Experimental group 2 (preheating for 10 minutes) Experimental group 3 (preheating for 15 minutes) Experimental group 4 (preheating 20 minutes) Ct value average Ct value average Ct value average Ct value average 6.02 X 10 5 CFU / mL 31.29 31.14 30.98 27.79 30.90 30.54 30.49 30.60 31.27 30.48 30.81 30.61 30.85 30.96 29.90 30.71 6.02 X 10 4 CFU / mL 33.31 33.44 33.59 33.44 33.83 33.91 32.94 33.44 33.06 33.27 33.09 33.85 33.96 33.45 34.82 33.54 6.02 X 10 3 CFU / mL 37.34 37.31 36.77 37.90 39.19 40.83 35.80 36.41 38.47 39.53 38.49 37.43 36.12 37.39 44.80 35.99

[0410] density Experimental group 1 (preheating 5 minutes) Experimental group 2 (preheating for 10 minutes) Experimental group 3 (preheating for 15 minutes) Experimental group 4 (preheating 20 minutes) Ct value average Ct value average Ct value average Ct value average 1.4 X 10 5 CFU / mL 33.76 33.44 32.87 27.79 33.32 32.60 32.72 32.78 33.49 32.44 32.55 32.71 33.08 32.56 31.93 32.90 1.4 X 10 4 CFU / mL 36.44 36.05 35.18 33.44 35.38 35.48 35.17 35.67 35.62 36.22 35.05 36.01 36.10 35.52 36.03 35.81 1.4 X 10 3 CFU / mL N / A - 41.81 - 39.14 38.45 N / A - 43.44 N / A 38.18 N / A N / A 38.03 38.02 40.73

[0411] density Experimental group 1 (preheating 5 minutes) Experimental group 2 (preheating for 10 minutes) Experimental group 3 (preheating for 15 minutes) Experimental group 4 (preheating 20 minutes) Ct value average Ct value average Ct value average Ct value average 1 X 10 4 CFU / mL 30.20 29.57 29.45 29.57 30.06 29.56 29.32 29.65 29.02 29.34 29.57 29.95 28.63 29.91 29.04 29.69 1 X 10 3 CFU / mL 31.53 32.35 34.13 32.58 31.87 32.29 32.86 33.21 33.11 31.25 31.82 33.13 32.40 32.37 33.16 33.64 1 X 10 2 CFU / mL 38.16 - 37.95 37.29 37.50 - 39.51 - N / A 37.75 N / A N / A N / A 36.15 N / A 34.60

[0412] As shown in Table 19 above, in the case of Streptococcus piogenes, experimental group 2, which was preheated for 10 minutes, showed overall lower Ct values.

[0413] In addition, as shown in Table 20, in the case of Streptococcus disgalactiae subspecies equisimilis, experimental group 2, which was preheated for 10 minutes, showed overall lower Ct values, and experimental groups 3 and 2 were able to detect small amounts of the strain.

[0414] In addition, as shown in Table 21, in the case of Arcanobacterium hamolitiscum, all experimental groups showed similar Ct values, and experimental group 2 was able to detect a small amount of the strain.

[0415] As mentioned above, in terms of the efficiency, sensitivity, and TAT of the nucleic acid amplification reaction, the optimal preheating time of the cell lysis composition was found to be 10 minutes.

[0416] Although preferred embodiments of the present invention have been described, it will be understood that modifications and variations falling within the spirit of the present invention will be obvious to those skilled in the art and that the scope of the present invention is determined by the appended claims and their equivalents.

Claims

Claim 1 A method for extracting nucleic acids from a biological sample in an automated liquid handling system comprising the following steps: (i) dispensing a cell lysis composition into an empty reaction vessel mounted on a heating element within the automated liquid handling system, wherein the cell lysis composition serves to lyse cells within the biological sample; (ii) pre-incubating the cell lysis composition at 50°C or higher by the heating element; (iii) dispensing the biological sample into the reaction vessel to prepare a mixture; and (iv) incubating the mixture at 50°C or higher by the heating element to extract nucleic acids from the biological sample, wherein steps (i)-(iv) are controlled by a controller within the automated liquid handling system, and steps (i) and (iii) are performed by a pipetting channel. Claim 2 A method according to claim 1, wherein the biological sample is a swab, saliva, or a mixture thereof from a subject. Claim 3 In claim 1, the biological sample is Streptococcus piogenes ( Streptococcus pyogenes ), Streptococcus equity subspecies equity ( Streptococcus equi subsp. Equi ) , Streptococcus equi subspecies zueepidemicus ( Streptococcus equi subsp. zooepidemicus ) , Streptococcus disgalactiae subspecies disgalactiae ( Streptococcus dysgalactiae subsp. dysgalactiae ) , Streptococcus disgalactiae subspecies equityimilis ( Streptococcus dysgalactiae subsp. equisimilis ) , Streptococcus cannis ( Streptococcus canis ) , Arcanobacterium hamolitiscum Arcanobacterium haemolyticum A method characterized by containing or suspected of containing ), and a combination thereof. Claim 4 A method according to claim 1, characterized in that the heating element serves to heat the reaction vessel mounted thereon to a predetermined temperature while shaking it. Claim 5 A method according to claim 1, characterized in that the reaction vessel is a deep-well plate or a well plate. Claim 6 A method according to claim 1, wherein the cell lysis composition comprises (a) guanidine hydrochloride (GuHCl); (b) cetyltrimethylammonium bromide (CTAB); and (c) polyethylene glycol (PEG) having a molecular weight of 200 to 1000 Da. Claim 7 A method according to claim 1, wherein the cell lysis composition comprises an amount of 30 to 300 mM of GuHCl, an amount of CTAB less than about 0.05 to about 0.5 weight% based on the total weight of the cell lysis composition, and an amount of PEG of about 5 to about 80 weight% based on the total weight of the cell lysis composition. Claim 8 A method according to claim 6, wherein the cell lysis composition further comprises a detergent, a chelating agent, a buffer, or a combination thereof. Claim 9 A method according to claim 1, characterized in that the cell lysis composition of step (i) is dispensed in an amount of 10 to 100 μL. Claim 10 A method according to claim 1, characterized in that the method further comprises the step of heating an empty reaction vessel by a heating element in an automated liquid handling system prior to step (i). Claim 11 A method according to claim 10, characterized in that the heating element of step (ii) is controlled by a controller so that its temperature increases to 95°C or higher. Claim 12 A method according to claim 1, characterized in that the heating element of step (ii) is controlled by a controller so that its temperature is maintained at 95°C or higher. Claim 13 A method according to claim 1, characterized in that the pre-incubation of step (ii) is performed for at least 5 minutes. Claim 14 A method according to claim 1, characterized in that the biological sample of step (iii) is dispensed in an amount of 10 to 100 μL. Claim 15 A method according to claim 1, characterized in that the biological sample of step (iii) is dispensed in an equal amount to the cell lysis composition dispensed in step (i). Claim 16 A method according to claim 1, characterized in that the heating element of step (iv) is controlled by a controller so that its temperature increases to 95°C or higher. Claim 17 A method according to claim 1, characterized in that the extracted nucleic acid is applied directly to a nucleic acid amplification reaction without further purification. Claim 18 A method according to claim 17, characterized in that the nucleic acid amplification reaction is PCR, real-time PCR, or LAMP (Loop Mediated Isothermal Amplification).

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