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

Abstract

The present disclosure relates to a method for extracting nucleic acids from a biological sample in an automated liquid handling system, and more particularly, to a method for extracting nucleic acids in large quantities and with improved sensitivity from a biological sample without purification using a cell lysis composition in an automated liquid handling system. The method of the present disclosure enables obtaining nucleic acids without complex purification step. Therefore, the method of the present disclosure does not require reagents and tools used for purifying nucleic acids, thereby reducing the cost and time for molecular diagnosis.

Description

METHOD FOR EXTRACTING NUCLEIC ACIDS FROM BIOLOGICAL SAMPLE IN AUTOMATED LIQUID HANDLING SYSTEM

[0001] Cross-Reference To Related Applications

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

[0003] Technical 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 particularly, to a method for extracting nucleic acids in large quantities and with improved sensitivity from a biological sample without purification using a cell lysis composition in an automated liquid handling system.

[0005]

[0006] Molecular diagnosis is the fastest growing segment in the in vitro diagnostic market for early diagnosis of diseases. In particular, nucleic acid-based methods are used to diagnose causative genetic factors associated with viral or bacterial infections with high specificity and sensitivity.

[0007] Most nucleic acid-based methods involves amplification of target nucleic acids (e.g., viral or bacterial nucleic acids). As a representative example, polymerase chain reaction (PCR) involves repeated cycles of denaturation of double-stranded DNA, annealing of primers to DNA templates, and extension of the primers by DNA polymerase (Mullis et al., U.S. Pat. Nos. 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.

[0008] The real-time PCR method comprises collecting a sample,e.g., nasopharyngeal, or oropharyngeal swab sample from a subject, storing it in a transport medium, and then extracting and purifying nucleic acids prior to the real-time PCR.

[0009] The extraction and purification of nucleic acids are critical for eliminating potential PCR inhibitors and ensuring reliable results. However, they are time-consuming, costly, and require skilled technicians for accurate execution, making them a bottleneck in the overall diagnostic testing workflow.

[0010] To address the problems, automated high throughput systems (Cameron, A. et al., J. Clin. Microbiol. 59, 2; Kim, T. Y.et al. Ann. Lab. Med. 42, 473-477; Ji, L.et al. Environ. Sci. Technol. 56, 13398-13407), and methods using magnetic beads (Pham, X. H.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), or a microfluidic device (Obino, D.et al. Sens. (Basel) 21, 3059; Jin, C. E.et al. Anal. Chem. 89, 7502-7510; Wen, J. et al., Anal. Chem. 80, 6472-6479) have been proposed.

[0011] In addition to these technical developments, efforts have been made to optimize protocols or introduce extraction-free methods (Fomsgaard, A. S. & Rosenstierne, M. W. Euro Surveill 25, 14; Villota, S. D.et al., J. Virol. Methods 298, 114302; Lownik, J. C., Way, G. W., Farrar, J. S. & Martin, R. K., J. Mol. Diagn. 23, 1671-1679; Kim, Y. K. & Chang, S. H., J. Virol. Methods 296, 114217; Visseaux, B.et al., J. Virol. Methods 291, 11408; Smyrlaki, I.et al., Nat. Commun. 11, 4812).

[0012] Extraction-free methods can simplify testing and facilitate processing by avoiding time-consuming and labor-intensive extraction steps. Examples thereof include diluting a sample 1:1, heating a sample at about 100℃, treating a sample with a lysis material, or using a combination thereof to reduce inhibitory substances that may interfere with PCR.

[0013] Despite these advantages, the extraction-free methods suffer from low sensitivity, due to the presence of PCR inhibitors and the degradation of nucleic acids in the sample.

[0014] There is a need in the art to develop a novel extraction-free method.

[0015] Throughout this specification, a number of patents and documents are referenced and their references are indicated in parentheses. In order to more clearly describe the present disclosure and the level of the technical field to which the present disclosure pertains, the disclosures of these patents and documents are incorporated herein by reference in their entirety.

[0016]

[0017] The present inventors have made extensive efforts to develop a novel method for extracting nucleic acids, which can reduce the time and labor required for nucleic acid extraction and improve low sensitivity.

[0018] As a result, the present inventors have found that nucleic acids can be effectively extracted without purification by pre-heating 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, and that the extracted nucleic acids can be used in nucleic acid amplification reactions with high sensitivity.

[0019] Accordingly, it is an object of the present invention to provide a method for extracting nucleic acids from a biological sample in an automated liquid handling system.

[0020] Other objects and advantages of the present disclosure will become more apparent from the following detailed description together with the appended claims.

[0021]

[0022] In an aspect of the present disclosure, there is provided a method for extracting nucleic acids from a biological sample in an automated liquid handling system, comprising:

[0023] (i) dispensing a cell lysis composition into an empty reaction vessel, mounted on a heating element in the automated liquid handling system, wherein the cell lysis composition serves to lyse cells in the biological sample;

[0024] (ii) pre-incubating the cell lysis composition at 50℃ or higher by the heating element;

[0025] (iii) dispensing the biological sample into the reaction vessel to prepare a mixture; and

[0026] (iv) incubating the mixture at 50℃ or higher by the heating element to extract nucleic acids from the biological sample;

[0027] wherein steps (i)-(iv) are controlled by a controller and steps (i) and (iii) are performed by means of pipetting channels in the automated liquid handling system.

[0028] In certain embodiments, the biological sample is a swab, saliva, or a mixture thereof from a subject.

[0029] In certain embodiments, the biological sample contains or is suspected of containingStreptococcus pyogenes,Streptococcus equi subsp. Equi,Streptococcus equi subsp. zooepidemicus,Streptococcus dysgalactiae subsp. dysgalactiae,Streptococcus dysgalactiae subsp. equisimilis,Streptococcus canis,Arcanobacterium haemolyticum, or combinations thereof.

[0030] In certain embodiments, the heating element serves to heat the reaction vessel mounted thereon to a predefined temperature while shaking.

[0031] In certain embodiments, the reaction vessel is a deep-well plate or a well plate.

[0032] In certain embodiments, 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.

[0033] In certain embodiments, the cell lysis composition comprises GuHCl in an amount of 30 to 300 mM, CTAB in an amount of 0.05 to less than 0.5 wt% based on the total weight of the cell lysis composition, and PEG in an amount of 5 to 80 wt% based on the total weight of the cell lysis composition.

[0034] In certain embodiments, the cell lysis composition further comprises a detergent, a chelating agent, a buffering agent, or combinations thereof.

[0035] In certain embodiments, the cell lysis composition of step (i) is dispensed in an amount of 10 to 100 μL.

[0036] In certain embodiments, the method further comprises heating the empty reaction vessel by the heating element in the automated liquid handling system, prior to step (i).

[0037] In certain embodiments, the heating element of step (ii) is controlled by the controller such that its temperature increases to 95℃ or higher.

[0038] In certain embodiments, the heating element of step (ii) is controlled by the controller such that its temperature remains at 95℃ or higher.

[0039] In certain embodiments, the pre-incubation of step (ii) is carried out for at least 5 minutes.

[0040] In certain embodiments, the biological sample of step (iii) is dispensed in an amount of 10 to 100 μL.

[0041] In certain embodiments, the biological sample of step (iii) is dispensed in an amount equal to that of the cell lysis composition dispensed in step (i).

[0042] In certain embodiments, the heating element of step (iv) is controlled by the controller such that its temperature increases to 95℃ or higher.

[0043] In certain embodiments, the extracted nucleic acid is directly applied to a nucleic acid amplification reaction without further purification.

[0044] In certain embodiments, the nucleic acid amplification reaction is PCR, real-time PCR, or Loop Mediated Isothermal Amplification (LAMP).

[0045]

[0046] The features and advantages of the present invention are summarized as follows:

[0047] (a) The method of the present disclosure enables obtaining nucleic acids without complex purification step. Therefore, the method of the present disclosure does not require reagents and tools used for purifying nucleic acids, thereby reducing the cost and time for molecular diagnosis.

[0048] (b) The method of the present disclosure enables extracting nucleic acids in large quantities by using an automated liquid handling system. Therefore, the method of the present disclosure can reduce human error and labor, and is useful in large hospitals, entrusted testing institutions, and research institutes.

[0049] (c) When using a reaction vessel with low thermal conductivity, such as a deep-well plate, in an automated liquid handling system, it is difficult to raise the temperature of the cell lysis composition or the mixture of the cell lysis composition and a sample in the reaction vessel to around 100℃. Therefore, conventional nucleic acid extraction method by heating cannot achieve high nucleic acid extraction efficiency. In contrast, the method of the present invention can achieve high nucleic acid extraction efficiency through pre-incubation of the cell lysis composition at 50℃ or higher, even without raising the temperature of the cell lysis composition or the mixture of the cell lysis composition and the sample to around 100℃.

[0050] (d) The method of the present invention can further improve extraction efficiency by using a cell lysis composition having a specific composition.

[0051]

[0052] The present inventors have made extensive efforts to develop a novel method for extracting nucleic acids, which can reduce the time and labor required for nucleic acid extraction and improve low sensitivity.

[0053] As a result, the present inventors have found that nucleic acids can be effectively extracted without purification by pre-heating 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, and that the extracted nucleic acids can be used in nucleic acid amplification reactions with high sensitivity.

[0054]

[0055] I. Definition

[0056] Hereinafter, the terms used in connection with the present invention are defined.

[0057] As used herein, the term "biological sample" refers to an analyte obtained from a subject, which contains or is suspected of containing nucleic acids 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, aspiration, milk, urine, feces, eye fluids, semen, brain extracts, spinal fluid, joint fluid, thymus fluid, bronchial lavage fluid, ascites, and amniotic fluid. In certain embodiments, the biological sample is a swab, saliva, or a mixture thereof from a subject. In certain embodiments, the swab includes, but not limited thereto, a throat swab or a nasal swab, such as nasopharyngeal swab. The sample may be used interchangeably with "specimen" herein.

[0058] As used herein, the term "subject" refers to an individual suspected of containing a target nucleic acid (e.g., from a specific pathogen) to be detected using the method of the present disclosure. Examples of the subject include, but are not limited to, mammals such as dogs, cats, rodents, primates, humans, and the like, and in particular, humans.

[0059] As used herein, the term "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" refers to a single- or double-stranded deoxyribonucleotide or ribonucleotide polymer, and the nucleotide may include a derivative of a natural nucleotide, a non-natural nucleotide, or a modified nucleotide, which can function in the same manner as a naturally occurring nucleotide.

[0060] As used herein, the term "target nucleic acid", "target nucleic acid sequence", or "target sequence" refers to a nucleic acid sequence whose presence is to be detected. The nucleic acid extracted by the method of the present disclosure may include a nucleic acid to be detected, such as a nucleic acid from a specific organism, particularly a pathogen, or a mutated nucleic acid, which is referred herein to as a target nucleic acid. The presence of the target nucleic acid may be determined by various methods known to those skilled in the art, such as nucleic acid amplification-based molecular diagnosis (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 of an organism, for example, a pathogen from which the target nucleic acid is derived, or whether there is mutation.

[0061] The present disclosure provides a method for obtaining nucleic acids in large quantities and in a simple manner for use in subsequent determination of the presence of target nucleic acids, such as molecular diagnosis and sequencing as described above.

[0062] As used herein, the term "automated liquid handling system" refers to a system that automatically performs repetitive liquid handling operations that were performed manually in a laboratory. The automated liquid handling system typically serves to dispense a predefined amount of liquid (e.g., reagents, samples, etc.) into a desired vessel, such as for pipetting, sample preparation, microplate cleaning, and the like. The automated liquid handling system has the advantages of reducing human error or operation time and costs and enabling high throughput.

[0063] Automated liquid handling systems are commercially available, examples of which include, but are not limited to, Microlab Prep, Microlab NIMBUS, Microlab STAR, and Microlab VANTAGE from Hamilton; 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 from Eppendorf; flowbot® ONE Liquid Handling Robot from Flow-Robotics; ASSIST PLUS Pipetting Robot, MIRO CANVAS NGS Prep System, and ASSIST Pipetting Platform from INTEGRA Biosiences; Opentrons Flex NGS Workstation and OT-2 Pipetting Robot from Opentrons; Biotage® Extrahera™ Automated Sample Processing System and Biotage® Extrahera™ LV-200 from Biotage; 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 Andrew+ Pipetting Robot from Waters Andrew Alliance.

[0064] It will be appreciated by those skilled in the art that the automated liquid handling system 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, and the like, which may be altered or adjusted depending upon the application. In addition to the above, additional components may be integrated into the automated liquid handling system if required.

[0065] The automated liquid handling system used in the method of the present disclosure includes a controller that serves to control the entire step, a pipetting channel that serves to draw and dispense a cell lysis composition and a biological sample, and a heating element to heat a liquid contained in a reaction vessel to a predefined temperature.

[0066] The term "automated liquid handling system" is used interchangeably with other terms known in the art, such as automated liquid handling / processing / pipetting / dispensing robot / station / platform, or similar terms.

[0067] As used herein, the term "reaction vessel" refers to a space in which a reaction for extracting nucleic acids from a sample is performed. The term may be used interchangeably with "container" or "carrier". The reaction vessel accommodates a substance such as a liquid sample or reagent.

[0068] As used herein, the term "cell lysis composition" or "composition for lysing cells" refers to a composition that serves to lyse cells of an organism that includes a nucleic acid (e.g., DNA or RNA) and release the nucleic acids. Specifically, the cell lysis composition destroys cells of an organism, such as an animal, a plant, a yeast, a bacterium, a virus, and the like, upon contact with a biological sample obtained from a subject, thereby releasing nucleic acids therein to the outside.

[0069] Conventionally, in order to isolate nucleic acids from cells of an organism present in a biological sample, a series of steps have been performed, including lysis of cells, binding of nucleic acids, washing of nucleic acids, purification of nucleic acids, and elution of nucleic acids. For optimized performance of each of the steps, several reagents have been developed, including, but not limited to, a lysis buffer, a binding buffer, a wash buffer, an elution buffer, and the like.

[0070] The "cell lysis composition" of the present disclosure is similar to a conventional lysis buffer in terms of its function to lyse cells. However, conventional lysis buffers contain components that inhibit nucleic acid amplification reactions such as PCR, so the lysate obtained using the buffer cannot be used in the reaction directly without purification. In contrast, the cell lysis composition according to the present disclosure does not contain components that inhibit the nucleic acid amplification reaction, so the lysate obtained using the composition can be used in the nucleic acid amplification reaction directly 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.

[0071] In addition, the "cell lysis composition" according to the present disclosure may also be referred to as an "extraction-free" composition, buffer, reagent, or solution, because it allows nucleic acids to be obtained without nucleic acid extraction, that is, nucleic acid purification.

[0072] The cell lysis composition described above does not contain any inhibitor of a nucleic acid amplification reaction.

[0073] As used herein, "cell lysis" or "lysis" means degradation or destruction of the outer boundary or cell membrane to release intracellular materials such as DNA, RNA, proteins, or organelles from the cell. In particular, cell lysis or lysis refers to degradation or destruction of cells to release nucleic acids,i.e., DNA or RNA, from the cells.

[0074] In the method of the present disclosure, lysis of cells in a biological sample occurs by the cell lysis composition described above.

[0075] The cells in the biological sample that can be lysed by the composition as described above may be derived from a variety of organisms, particularly pathogens, such as Gram-positive bacteria.

[0076] In certain embodiments, the biological sample contains or is suspected of containingStreptococcus pyogenes,Streptococcus equi subsp. Equi,Streptococcus equi subsp. zooepidemicus,Streptococcus dysgalactiae subsp. dysgalactiae,Streptococcus dysgalactiae subsp. equisimilis,Streptococcus canis,Arcanobacterium haemolyticum, or combinations thereof.

[0077] In certain embodiments, the cells in the biological sample are derived from any one or more bacteria selected from the group consisting ofStreptococcus pyogenes,Streptococcus equi subsp. Equi,Streptococcus equi subsp. zooepidemicus,Streptococcus dysgalactiae subsp. dysgalactiae,Streptococcus dysgalactiae subsp. equisimilis,Streptococcus canis, andArcanobacterium haemolyticum.

[0078] The cell lysis composition according to the present disclosure is used to isolate nucleic acids from a biological sample obtained from a subject that is suffering from or is likely to suffer from pharyngitis in order to determine the presence of causative pathogens in the subject.

[0079] Pharyngitis is a disease that causes inflammation in the pharynx along with neck pain and is transmitted through droplets, coughing, sneezing, etc. Pharyngitis itself is not fatal, but without rapid antibiotic treatment, it may cause various complications such as septic pharyngitis and streptococcal toxic shock syndrome. It is required to accurately detect the causative agent in order to minimize complications caused by pharyngitis and to reduce the use of unnecessary antibiotics.

[0080] The major causative agents of the pharyngitis are β-hemolyticStreptococcus,Arcanobacterium haemolyticum, and the like, and examples of the β-hemolyticStreptococcusincludeStreptococcus pyogenesbelonging to the group A Streptococcus (GAS), andStreptococcus dysgalactiae subsp. equisimilisandStreptococcus dysgalactiae subsp. dysgalactiaebelonging to the group C Streptococcus (GCS) or the group G Streptococcus (GGS).

[0081] The GAS is a Gram-positive bacterium of the genusStreptococcusthat exhibits β-hemolysis and has the A, C and G antigens according to the Lancefield classification.

[0082] The GCS or GGS exhibits β-hemolysis and is a Gram-positive bacterium of the genusStreptococcus, which has C and G antigens according to the Lancefield classification.

[0083] Subjects diagnosed as having GAS, GCS or GGS are treated with antibiotics such as penicillin, amoxillin, or benzathine, and if allergic to penicillin, they are treated with antibiotics such as cephalexin, cepadroxyl, clindamycin, azithromycin, or clarithromycin.

[0084] Meanwhile,Arcanobacterium hamolyticumis a species of bacteria classified as Gram-positiveBacillus. Subjects diagnosed as havingArcanobacterium haemolyticumare treated with an antibiotic such as erythromycin, azithromycin, gentamicin, or clindamycin.

[0085] Although the prescription for GAS, GCS or GGS is different from that forArcanobacterium hamolyticum, the symptoms caused by GAS, GCS or GGS infection are similar to those caused byArcanobacterium hamolyticuminfection, making it difficult to distinguish them from each other. Thus, discrimination between them helps prevent abuse of antibiotics such as penicillin and prescribe precise treatments.

[0086] The cell lysis composition according to the present disclosure may be used to prepare a sample for a nucleic acid amplification reaction,e.g., real-time PCR, for detecting causative agents in patients with pharyngitis. In order to perform a nucleic acid amplification reaction, a complicated process of extracting and purifying nucleic acids from a biological sample is conventionally required. However, the method according to the present disclosure allows a nucleic acid to be separated from a biological sample only by a simple incubation, and thus a lysate obtained by the method according to the present disclosure can be applied directly to a nucleic acid amplification reaction.

[0087] In certain embodiments, the nucleic acid obtained by the method of the present disclosure may be mixed with a nucleic acid amplification reagent and subjected to a nucleic acid amplification reaction.

[0088] 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 the amplification reaction.

[0089] In certain embodiments, the nucleic acid amplification reaction include, but is not limited to, PCR, real-time PCR, and loop mediated isothermal amplification (LAMP).

[0090] Specifically, amplification is performed according to polymerase chain reaction (PCR), which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159. Other examples include ligase chain reaction (LCR) (U.S. Pat. Nos. 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)), 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., 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).

[0091] In an embodiment, the amplification of the target nucleic acid may be performed by a nucleic acid amplification method, for example, PCR, which involves a change in temperature. In the nucleic acid amplification method, several or dozens of cycles including denaturing step, primer annealing step, and extension (or amplification) step may be repeated.

[0092] In an embodiment, the amplification of the target nucleic acid may be performed by a nucleic acid amplification method that does not involve a change in temperature, that is, an isothermal amplification method. Examples of isothermal amplification methods include, but are not limited to, Rolling Circle Amplification (RCA, M. M. Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, J. A. Ankrum, X. C. Le and 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), Nucleic Acid Sequence-Based Amplification (NASBA, A. Borst, J. Verhoef, E. Boel and A. C. Fluit, Clin. Lab., 2002, 48, 487-492), Strand Displacement Amplification (SDA, B. J. Toley, I. Covelli, Y. Belousov, S. Ramachandran, E. Kline, N. Scarr, N. Vermeulen, W. Mahoney, B. R. 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).

[0093] Although PCR is mainly described as an example of a target nucleic acid amplification method throughout the specification, it will be appreciated by those skilled in the art that other nucleic acid amplification reactions may be used.

[0094] According to an embodiment of the present disclosure, when the target nucleic acid is RNA, the amplification reaction may include reverse transcription. Details thereof are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Noonan, K.F. et al., Nucleic Acids Res. 16:10366 (1988).

[0095] The nucleic acid amplification reagent used in the method of the present disclosure comprises a set of oligonucleotides for detecting a target nucleic acid.

[0096] The set of oligonucleotides used in the method of the present disclosure comprises: (i) an amplifying oligonucleotide serving to amplify a target nucleic acid; and (ii) a signaling oligonucleotide having a fluorescent label linked thereto, serving to generate a signal in the presence of a target nucleic acid, wherein the amplifying oligonucleotide and the signaling oligonucleotide are the same or different.

[0097] In one embodiment, the amplifying oligonucleotide is a "primer" known in the art. As used herein, the term "primer" refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced,i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact length of the primers will depend on many factors, including temperature, application, and source of primer.

[0098] The primer may include a forward primer (also referred to as an upstream primer or an upstream oligonucleotide), a reverse primer (also referred to as a downstream primer or a downstream oligonucleotide), or both. The amplifying oligonucleotide may be an oligonucleotide having a structure known in the art and may be synthesized in a manner known in the art.

[0099] As used herein, the term "signaling oligonucleotide" refers to an oligonucleotide involved in the generation of the signal being detected. According to one embodiment, the signaling oligonucleotide comprises an oligonucleotide involved in actual signal generation. For example, hybridization or non-hybridization of signaling oligonucleotides with other oligonucleotides (e.g., oligonucleotides comprising nucleotide sequences complementary to target nucleic acids or signaling oligonucleotides) determines signal generation.

[0100] In one embodiment, the signaling oligonucleotide is a 'probe' known in the art. As used herein, the term "probe" refers to a single-stranded nucleic acid molecule comprising portion or portions substantially complementary to a target nucleic acid. According to one embodiment, the 3'-end of the probe is "blocked" to prevent its extension. The blocking may be achieved in accordance with conventional methods. For instance, the blocking may be performed by adding to the 3'-hydroxyl group of the last nucleotide a chemical moiety such as biotin, labels, a phosphate group, alkyl group, non-nucleotide linker, phosphorothioate or alkane-diol. Alternatively, the blocking may be carried out by removing the 3'-hydroxyl group of the last nucleotide or using a nucleotide with no 3'-hydroxyl group such as dideoxynucleotide.

[0101] In one embodiment, the signaling oligonucleotide has at least one fluorescent label linked thereto, wherein the at least one fluorescent label in the signaling oligonucleotide generates a fluorescent signal.

[0102] The set of oligonucleotides including the signaling oligonucleotide may generate a signal by one of methods known in the art.

[0103] In one embodiment, the set of oligonucleotides generates a signal by formation of a duplex from the signaling oligonucleotide or dissociation of the duplex. Particularly, a signal is generated by the formation of a duplex between a target nucleic acid and a signaling oligonucleotide specifically hybridized with the target nucleic acid.

[0104] The signal by the formation of a duplex between a target nucleic acid and a signaling oligonucleotide may be generated by various methods, including Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), Sunrise (or Amplifluor) method (Nazarenko et al,, Nucleic Acids Research, 25(12):2516-2521 (1997), and U.S. Pat. No. 6,117,635), Lux method (U.S. Pat. No. 7,537,886), Plexor method (Sherrill C B, et al., Journal of the American Chemical Society, 126:4550-45569 (2004)), Molecular Beacon method (Tyagi et al., Nature Biotechnology v.14 MARCH 1996), HyBeacon method (French DJ et al., Mol. Cell Probes, 15(6):363-374(2001)), adjacent hybridization probe method (Bernard, P.S. et al., Anal. Biochem., 273:221(1999)) and LNA method (U.S. Pat. No. 6,977,295).

[0105] In one embodiment, the set of oligonucleotides generates a signal by cleavage of a mediation oligonucleotide hybridized to the target nucleic acid and subsequent formation or dissociation of a duplex between the cleavage product and the signaling oligonucleotide, which occurs dependently on the presence of the target nucleic acid.

[0106] The signal by the duplex formed in a dependent manner on cleavage of the mediation oligonucleotide may be generated by various methods, including PTOCE (PTO cleavage and extension) method (WO 2012 / 096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013 / 115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (WO 2014 / 104818), methods described in U.S. Pat. Nos. 11,034,997, 11,028,433, and 10,590,469, and U.S. Pat. Appl. No. 2020-0048682.

[0107] In one embodiment, the set of oligonucleotides generates a signal by hybridization of the signaling oligonucleotide with a target nucleic acid and subsequent cleavage of the signaling oligonucleotide, which occurs dependently on the presence of the target nucleic acid.

[0108] The signal by hybridization of the signaling oligonucleotide with a target nucleic acid and subsequent cleavage of the signaling oligonucleotide may be generated by various methods, including TaqMan probe method (U.S. Pat. Nos. 5,210,015 and 5,538,848).

[0109] In one embodiment, the set of oligonucleotides generates a signal by cleavage of the signaling oligonucleotide in a dependent manner on cleavage of the mediation oligonucleotide that is specifically hybridized to the target nucleic acid.

[0110] The signal by cleavage of the signaling oligonucleotide in a dependent manner on cleavage of the mediation oligonucleotide may be generated by various methods, including Invader assay (U.S. Pat. No. 5,691,142), PCEC (PTO Cleavage and Extension-Dependent Cleavage) method (WO 2012 / 134195) and a method described in U.S. Pat. No. 7,309,573.

[0111] 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 a single fluorescent label, interactive dual fluorescent labels and interactive triple labels.

[0112] In one embodiment, the single label provides different signal intensities depending on its presence on a double strand or single strand. The preferable types and binding sites of single fluorescent labels used in the method of the present disclosure are disclosed U.S. Pat. Nos. 7,537,886 and 7,348,141, the teachings of which are incorporated herein by reference in their entirety. For example, the single fluorescent label includes JOE, FAM, TAMRA, ROX, and fluorescein-based label. The single label may be linked to oligonucleotides by various methods. For instance, the label is linked to probes through a spacer containing carbon atoms (e.g., 3-carbon spacer, 6-carbon spacer or 12-carbon spacer).

[0113]

[0114] II. Method for extracting nucleic acids

[0115] As described above, the present disclosure provides a method for extracting nucleic acids from a biological sample without purification of the nucleic acids by incubation of the biological sample with a cell lysis composition.

[0116] In addition, the present disclosure provides a method for extracting nucleic acids in large quantities and conveniently using an automated liquid handling system.

[0117] Furthermore, the present disclosure provides a method for achieving high efficiency of nucleic acid extraction by increasing the temperature of a liquid in a reaction vessel to only about 50-60℃.

[0118] Specifically, the present disclosure provides a method for extracting nucleic acids from a biological sample in an automated liquid handling system, comprising:

[0119] (i) dispensing a cell lysis composition into an empty reaction vessel, mounted on a heating element in the automated liquid handling system, wherein the cell lysis composition serves to lyse cells in the biological sample;

[0120] (ii) pre-incubating the cell lysis composition at 50℃ or higher by the heating element;

[0121] (iii) dispensing the biological sample into the reaction vessel to prepare a mixture; and

[0122] (iv) incubating the mixture at 50℃ or higher by the heating element to extract nucleic acids from the biological sample;

[0123] wherein steps (i)-(iv) are controlled by a controller and steps (i) and (iii) are performed by means of pipetting channels in the automated liquid handling system.

[0124]

[0125] Hereinafter, the method of the present disclosure will be detailed.

[0126] Step (i): Dispensing a cell lysis composition

[0127] In step (i), a cell lysis composition is dispensed into an empty reaction vessel, mounted on a heating element in the automated liquid handling system.

[0128] Step (i) is controlled by a controller in the automated liquid handling system. Further, step (i) is performed by means of pipetting channels in the automated liquid handling system.

[0129] The description of the automated liquid handling system is found elsewhere herein. Examples of the automated liquid handling system available in the art include, but not limited to, Microlab STARlet, Microlab STAR, and Microlab NIMBUS from Hamilton; and Seegene STARlet or STARlet-AIOS from Seegene.

[0130] The heating element described above is installed within the automated liquid handling system.

[0131] As used herein, the term "heating element" refers to an apparatus that serves to raise the temperature of an object in contact. The heating element herein serves to raise the temperature of the reaction vessel placed thereon or a substance in the reaction vessel, such as the cell lysis composition or a mixture of the cell lysis composition and the biological sample.

[0132] In one embodiment, the heating element serves to heat the reaction vessel mounted thereon to a predefined temperature

[0133] In one embodiment, the heating element serves to heat the reaction vessel mounted thereon to a predefined temperature while shaking. The term "heating element" may be used interchangeably with various terms known in the art, such as a heating unit, a heating module, a heating component, a heating member, a heating means, a heater, a heater shaker, and the like.

[0134] The heating element may have a size capable of covering the entire reaction vessel. As an example, the heating element may be 100-200 mm long, 50-150 mm wide and 70-150 mm high, more particularly 150 mm long, 105 mm wide and 90 mm high.

[0135] The heating element may have a heating range from room temperature (e.g., about 25℃) to above 100℃. As an example, the heating element may heat up to a predefined temperature of 105℃.

[0136] However, the predefined temperature of the heating element and the temperature of the liquid in the reaction vessel may have a significant difference. This is because it is difficult for the liquid in the reaction vessel to rise to the predefined temperature of the heating element due to various factors such as low thermal conductivity of the reaction vessel or the liquid inside the reaction vessel. For example, even if the heating element is intended to rise to a fairly high temperature, such as 100℃, the liquid within a deep-well plate as a reaction vessel may only be at a temperature of 55℃ to 60℃.

[0137] It should be noted that although the predefined temperature of the heating element and the temperature of the liquid, such as the cell lysis composition or the mixture of the composition and the biological sample, in the reaction vessel are described in the specification, the temperature of the composition or the temperature of the mixture of the composition and the biological sample is a critical factor in achieving the purpose of the present disclosure. The heating elements of the present disclosure is freely adjusted to achieve a target temperature of the composition or mixture. For example, the predefined temperature of the heating element to raise the liquid in the reaction vessel having low thermal conductivity to a particular temperature will be higher than that of the heating element to raise the liquid in the reaction vessel having high thermal conductivity to the same temperature. One of ordinary skill in the art will be able to readily determine the predefined temperature of the heating element to raise the liquid in the reaction vessel to a particular temperature, depending on the type of the reaction vessel.

[0138] The heating element according to the present disclosure can shake a reaction vessel at various rates. As an example, the heating element may shake the reaction vessel at a rate of 200 to 3000 rpm, such as 2500 rpm.

[0139] The heating element according to the present disclosure may comprise an adapter as an auxiliary means for mounting the reaction vessel. The adapter may vary depending on the reaction vessel to which it is mounted.

[0140] In addition, the heating element according to the present disclosure may further comprise cooling means for lowering the temperature.

[0141] Examples of the heating element available in the art include, but not limited to, a heater shaker from Hamilton.

[0142] The reaction vessel according to the present disclosure is mounted on the heating element in the automated liquid handling system.

[0143] The reaction vessel is an empty reaction vessel that is not filled with a liquid.

[0144] 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.

[0145] 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.

[0146] In certain embodiments, the reaction vessel according to the present disclosure is a deep-well plate.

[0147] In one embodiment, the deep-well plate is made from 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.

[0148] The deep-well plate may be those made by various manufacturers, including, but is not limited to, Hamilton, Thermo Fisher Scientific, Axygen, and Corning.

[0149]

[0150] As describe above, the cell lysis composition dispensed into the reaction vessel in step (i) may be a direct lysis buffer or an extraction-free buffer known in the art. As described elsewhere herein, the cell lysis composition is used to obtain nucleic acids that will be applied without further purification directly to a subsequent reaction, so that it should not comprise components that inhibit subsequent amplification reactions, such as real-time PCR.

[0151] In certain embodiment, the cell lysis composition comprises:

[0152] (a) guanidine hydrochloride (GuHCl);

[0153] (b) cetyltrimethylammonium bromide (CTAB); and

[0154] (c) polyethylene glycol (PEG) having a molecular weight of 200 to 1000 Da.

[0155] The cell lysis composition according to the present disclosure has a combination of components and their optimal concentrations, which does not inhibit a nucleic acid amplification reaction and has an excellent cell lysis effect.

[0156]

[0157] Hereinafter, each component included in the composition of the present invention and the optimum concentration thereof will be described.

[0158] Guanidine hydrochloride (GuHCl)

[0159] Guanidine hydrochloride (GuHCl), also referred to as guanidinium chloride (GdmCl), is a strong chaotrope that can disrupt the hydrogen bonding network between water molecules. GuHCl is one of the strongest denaturants used in the physiochemical studies of protein folding. It also has the ability to decrease enzymatic activity and increase the solubility of hydrophobic molecules.

[0160] In the method according to the present disclosure, GuHCl serves to denature proteins and to lyse cells.

[0161] The GuHCl may be included in the composition in an amount of about 30 to about 300 mM. According to the Examples of present disclosure, 500 mM or more of GuHCl exhibits an inhibitory activity against a nucleic acid amplification reaction.

[0162] 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 therebetween.

[0163] In certain embodiments, the GuHCl is included in the composition in an amount of about 100 mM.

[0164] The GuHCl may be included in an amount of about 0.3 to about 3 wt%, based on the total weight of the composition.

[0165] In an embodiment, the GuHCl is included 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%, 2.5 to 3.0 wt%, or any value therebetween, based on the total weight of the composition.

[0166] In certain embodiments, the GuHCl is included in an amount of about 1.0 wt%, based on the total weight of the composition.

[0167] The lysates obtained using a composition comprising a combination of CTAB and PEG as described below exhibit a reduced fluorescence signal,i.e., reduced RFU value, in a nucleic acid reaction, whereas adding the GuHCl to the composition comprising the combination of CTAB and PEG can prevent the reduction in the fluorescence signal.

[0168]

[0169] Cetyltrimethylammonium bromide (CTAB)

[0170] Cetyltrimethylammonium bromide (CTAB), also referred to as hexadecyltrimethylammonium bromide, has a chemical formula of [(C16H33)N(CH3)3]Br. CTAB is a cationic surfactant, and the positive charge of CTAB binds electrostatically with negatively charged teichoic acid present in Gram-positive cells to create stress on the cells, thereby lysing the cells.

[0171] CTAB serves to maintain the integrity of the precipitated DNA during the isolation of the nucleic acids. Cells typically have high concentrations of macromolecules, such as glycoproteins and polysaccharides, which precipitate with the DNA during the extraction process, lowering the purity of the extracted DNA. The positive charge of CTAB denatures these molecules that would interfere with the isolation of these nucleic acids, helping to increase the purity of the DNA.

[0172] The CTAB may be included in an amount of about 0.05 to less than about 0.5 wt%, based on the total weight of the composition. According to the Examples of the present disclosure, 0.5 wt% or more of CTAB exhibits an inhibitory activity against a nucleic acid amplification reaction.

[0173] In an embodiment, the CTAB is included in an amount of 0.05 to 0.1 wt%, 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 therebetween, based on the total weight of the composition.

[0174] In certain embodiments, the CTAB is included in an amount of about 0.1 wt%, based on the total weight of the composition.

[0175] The lysates obtained using a composition comprising CTAB exhibit low Ct values,i.e., high performance in a nucleic acid amplification reaction.

[0176]

[0177] Polyethylene glycol (PEG)

[0178] Polyethylene glycol (PEG) has a chemical formula of H-(O-CH2-CH2)n-OH and is prepared by polymerization of ethylene oxide. PEG is a hydrophilic compound and acts as a chaotrope by binding to water molecules, leading to lysis of cells.

[0179] The PEG may be included in an amount of about 5 to about 80 wt%, based on the total weight of the composition. According to the Examples of the present disclosure, the PEG does not exhibit inhibitory activity against the nucleic acid amplification reaction even when it is added at a high concentration.

[0180] In an embodiment, the PEG is included in an amount of 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%, 30 to 70 wt%, 30 to 80 wt%, 40 to 50 wt%, 40 to 60 wt%, 40 to 70 wt%, 40 to 80 wt%, 50 to 60 wt%, 50 to 70 wt%, 50 to 80 wt%, 60 to 70 wt%, 60 to 80 wt%, 70 to 80 wt%, or any value therebetween, based on the total weight of the composition.

[0181] In certain embodiments, the PEG is included in an amount of 60 wt%, based on the total weight of the composition.

[0182] The PEG may have various molecular weights known in the art, for example, a molecular weight of 300 to 10,000,000 Da (g / mol).

[0183] In certain embodiments, the PEG has a molecular weight of 200 to 1,000 Da.

[0184] 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.

[0185] In one embodiment, the PEG is PEG300. The PEG300 has an average molecular weight of about 290 to about 305 Da and is a substantially colorless, transparent liquid.

[0186] 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.

[0187] In one embodiment, the PEG is PEG600. The PEG600 has an average molecular weight of about 550 to about 650 Da and is an almost white or colorless powder.

[0188] In one embodiment, the PEG is PEG900. The PEG900 has an average molecular weight of about 850 to about 950 Da and is an almost white or colorless powder.

[0189] In one embodiment, the PEG is PEG1000. The PEG1000 has an average molecular weight of about 900 to about 1,100 Da and is an almost white or colorless powder.

[0190] The lysates obtained using a compositions comprising PEG exhibit low Ct values and high RFU values,i.e., high performance in a nucleic acid amplification.

[0191]

[0192] As described above, the cell lysis composition according to the present disclosure comprises a combination of GuHCl, CTAB, and PEG.

[0193] The isolation and amplification of nucleic acids using a cell lysis composition comprising CTAB and PEG show decreased Ct values,i.e., improved detection performance for the target nucleic acid, as compared to conventional extraction and amplification of nucleic acids.

[0194] In addition, the isolation and amplification of nucleic acids using a cell lysis composition comprising a combination of GuHCl, CTAB, and PEG show higher RFU values,i.e., improved detection performance for the target nucleic acid, as compared to that using a cell lysis composition comprising CTAB and PEG.

[0195] Thus, the cell lysis composition comprising a combination of GuHCl, CTAB, and PEG can be used to detect a target nucleic acid with higher RFU values and lower Ct values.

[0196] In certain embodiments, the cell lysis composition comprises GuHCl in an amount of 30 to 300 mM, CTAB in an amount of 0.05 to less than 0.5 wt% based on the total weight of the cell lysis composition, and PEG in an amount of 5 to 80 wt% based on the total weight of the cell lysis composition.

[0197] In certain embodiments, the cell lysis composition according to the present disclosure comprises:

[0198] (a) about 100 mM of GuHCl;

[0199] (b) about 0.1 wt% of CTAB; and

[0200] (c) about 60 wt% of PEG200.

[0201]

[0202] Detergent

[0203] The cell lysis composition according to the present disclosure may further comprises a cationic, anionic, or nonionic detergent.

[0204] In an embodiment, the cationic detergent is selected from the group consisting of benzalkonium chloride (BAC), benzethonium chloride (BEC), methylbenzethonium, and cetylpyridinium chloride.

[0205] In one embodiment, the anionic detergent is n-lauroylsarcosine (NLS).

[0206] NLS, also referred to as sodium lauroyl sarcosinate or N-dodecanoyl-N-methylglycine sodium salt, has a chemical formula of CH3(CH2)10CON(CH3)CH2COONa. NLS acts on cell membranes and proteins to lyse cells.

[0207] The NLS may be included in an amount of about 0.05 to less than about 1 wt%, based on the total weight of the composition. According to the Examples of the present disclosure, 1 wt% or more of NLS exhibits inhibitory activity against a nucleic acid amplification reaction.

[0208] In an embodiment, the NLS is included 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 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%, 0.6 to 0.9 wt%, 0.6 to 0.8 wt%, 0.6 to 0.7 wt%, 0.7 to 0.9 wt%, 0.7 to 0.8 wt%, 0.8 to 0.9 wt%, or any value therebetween, based on the total weight of the composition.

[0209] In certain embodiments, the NLS is included in an amount of 0.05% by weight, based on the total weight of the composition.

[0210] In an embodiment, the non-ionic detergent is polyoxyethylene sorbitan monolaurate (Tween 20).

[0211] Tween 20, also referred to as polysorbate 20 or PEG (20) sorbitan monolaurate, has a chemical formula of C58H114O26. Tween 20 acts on cell membranes and proteins to lyse cells.

[0212] The Tween 20 may be included in an amount of about 5 to about 15 wt%, based on the total weight of the composition. According to the Examples of the present disclosure, 20 wt% or more of Tween 20 exhibits inhibitory activity against a nucleic acid amplification reaction.

[0213] In an embodiment, the Tween 20 is included in an amount of 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 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%, 12 to 13 wt%, 12 to 14 wt%, 12 to 15 wt%, 13 to 14 wt%, 13 to 15 wt%, 14 to 15 wt%, or any value therebetween, based on the total weight of the composition.

[0214] In certain embodiments, the Tween 20 is included in an amount of 5 wt%, based on the total weight of the composition.

[0215]

[0216] Chelating agent

[0217] The cell lysis composition according to the present disclosure may further include a chelating agent.

[0218] In an embodiment, the chelating agent includes ethylenediaminetetraacetic acid (EDTA).

[0219] EDTA is a metal chelating agent that chelates a divalent cation, such as a magnesium, zinc, manganese, nickel, or copper ion which is a cofactor of many enzymes such as DNase and proteases. EDTA serves to chelate cofactors of these enzymes, thereby deactivating these enzymes and preventing the degradation of nucleic acids. Additionally, EDTA serves to aid in the dissociation of the 40S and 60S ribosomal subunits.

[0220] The EDTA may be included in the composition in an amount of about 0.1 to about 10 mM.

[0221] In an embodiment, the EDTA is included 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 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 therebetween.

[0222] In certain embodiments, the EDTA is included in the composition in an amount of about 1 mM.

[0223] The EDTA may be included in an amount of about 0.02 to about 2 wt%, based on the total weight of the composition.

[0224] In an embodiment, the EDTA is included 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 1.6 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 therebetween, based on the total weight of the composition.

[0225]

[0226] Buffer

[0227] The cell lysis composition according to the present disclosure may further comprise a buffer.

[0228] In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris).

[0229] Tris, also referred to as trisamine, promethamol, tribase, Trizma, or THAM, is a primary amine having a chemical formula of (HOCH2)3CNH2. Tris serves to prevent a radical change in pH by maintaining a constant hydrogen ion concentration.

[0230] The Tris may be included in the composition in an amount of about 1 to about 50 mM.

[0231] In an embodiment, the Tris is included in an amount of 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 therebetween.

[0232] In certain embodiments, the Tris is included in the composition in an amount of about 10 mM.

[0233] The Tris may be included in an amount of about 0.01 to about 0.5 wt%, based on the total weight of the composition.

[0234] In an embodiment, the Tris is contained in an amount of 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 0.50 wt%, 0.40 to 0.45 wt%, 0.40 to 0.50 wt%, 0.45 to 0.50 wt%, or any value therebetween, based on the total weight of the composition.

[0235] In certain embodiments, the Tris is included in an amount of about 0.1 wt%, based on the total weight of the composition.

[0236]

[0237] 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 cell lysis and nucleic acid amplification reactions. Those skilled in the art can suitably adjust the pH of the cell lysis composition as long as it does not significantly affect cell lysis and nucleic acid amplification reactions. In one embodiment, the cell lysis composition has a pH of about 8. In other embodiments, the cell lysis composition has a pH of 8 to pH 12,e.g., pH 8, pH 9, pH 10, pH 11, or pH 12.

[0238]

[0239] In certain embodiments, the cell lysis composition according to the present disclosure comprises:

[0240] (a) about 100 mM of GuHCl;

[0241] (b) about 0.1 wt% of CTAB;

[0242] (c) about 60 wt% of PEG200;

[0243] (d) about 0.05 wt% of NLS;

[0244] (e) about 5 wt% of Tween 20;

[0245] (f) about 1 mM of EDTA; and

[0246] (g) about 10 mM of Tris,

[0247] wherein the composition has a pH of about 8.

[0248] Those skilled in the art will appreciate that even a slight change in the optimum concentration of each component constituting the cell lysis composition may have a similar performance.

[0249]

[0250] In one embodiment, the cell lysis composition described above in step (i) is dispensed in an amount of 10 to 100 μL. In certain embodiments, the cell lysis composition is dispensed in an amount of 30 μL, 40 μL, 50 μL, 60 μL, or 70 μL. In certain embodiments, the cell lysis composition is dispensed in an amount of 50 μL. The dispensed amount is an amount per reaction vessel, for example, when the reaction vessel is a 96 deep-well plate, 50 μL of the cell lysis composition may be dispensed into each well.

[0251]

[0252] In an embodiment, the method according to the present disclosure further comprises heating the empty reaction vessel by the heating element in the automated liquid handling system prior to step (i) above. Pre-heating the empty reaction vessel as described above may help to rapidly increase the temperature of the cell lysis composition to be filled in the empty reaction vessel in step (i).

[0253] In one embodiment, the heating element is controlled by a controller such that its temperature increases to 95℃ or higher. In certain embodiments, the temperature of the heating element may be heated to a predefined temperature such as 95℃, 100℃, or 105℃.

[0254] The time for heating is not particularly limited, and may be, for example, 1 minute 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.

[0255] The heating may be performed as soon as the empty reaction vessel is mounted on the heating element, or may be performed as soon as the automated liquid handling system is powered on.

[0256]

[0257] Step (ii): Pre-incubation of the cell lysis composition

[0258] In step (ii), the cell lysis composition is pre-incubated at 50℃ or higher by the heating element.

[0259] Step (ii) is controlled by a controller in the automated liquid handling system.

[0260]

[0261] According to the Examples of the present disclosure, it was found that the heating element in the automated liquid handling system has limitations in heating the liquid in the reaction vessel. Specifically, it was found that raising the heating element to a fairly high temperature such as 100-105℃ could not raise the temperature of the liquid, e.g., the cell lysis composition, in a reaction vessel such as a deep-well plate above 55-60℃. When a nucleic acid is intended to be extracted without purification using the 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 about 100℃.

[0262] The inventors have found that pre-incubation,e.g., pre-heating, of the cell lysis composition only prior to mixing the cell lysis composition with the biological sample has improved extraction efficiency over pre-incubation,e.g., pre-heating, of the biological sample only.

[0263] It is expected that pre-incubation of the biological sample results in evaporation of liquid components and thus concentration of PCR inhibitors (if present) in the biological sample, whereas pre-incubation of the cell lysis composition does not result in concentration of PCR inhibitors due to high boiling points such as 100℃ or higher of most of the chemicals constituting the cell lysis composition.

[0264]

[0265] In one embodiment, the cell lysis composition is incubated at about 55-65℃. For this, the heating element may be controlled by a controller such that its temperature increases to 95℃ or higher. The increase in the temperature of the heating element to 95℃ or higher can ensure that the temperature of the cell lysis composition within the reaction vessel is maintained at a temperature of 55℃ to 65℃.

[0266] In certain embodiments, the heating element may be controlled by a controller such that its temperature increases to 100℃. In certain embodiments, the heating element may be controlled by a controller such that its temperature increases to 105℃.

[0267] 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 therebetween. In certain embodiments, the pre-incubation in step (ii) is carried out for about 10 minutes.

[0268] The time for pre-incubation affects the efficiency and sensitivity of subsequent nucleic acid amplification reactions. Considering the improvement of efficiency and sensitivity of nucleic acid amplification reactions, the optimal time for pre-incubation of the cell lysis composition is about 10 minutes.

[0269]

[0270] Step (iii): Dispensing of the biological sample

[0271] In step (iii), the biological sample is dispensed into the reaction vessel to prepare a mixture.

[0272] Step (iii) is controlled by a controller in the automated liquid handling system. Step (iii) is also performed by means of pipetting channels in the automated liquid handling system.

[0273] The cell lysis composition pre-incubated prior to step (iii) is mixed with the biological sample. The mixing lyses the cells in the biological sample and releases the nucleic acids present in the cells out of the cells.

[0274] The 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 cell lysis composition described above, or (ii) directly mixing the biological sample with the cell lysis composition.

[0275] In the case of mixing a biological sample immersed in a transport medium with the cell lysis composition, the collected biological sample may be immersed in the transport medium, and a portion of the transport medium including the biological sample and the cell lysis composition may be mixed. The transport medium refers to a medium that can maintain the integrity of a sample, for example, cell integrity of a virus or bacteria.

[0276] 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.

[0277] 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 phosphate buffered saline (PBS) or normal saline.

[0278] The transport medium may not include a cell lysis component. In addition, the transport medium may be a liquid medium.

[0279] According to an embodiment, the transport medium containing the cell lysis composition may be mixed with the 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 ratio can improve the isolation of nucleic acids using the cell lysis composition and the amplification performance of the isolated nucleic acids.

[0280] In one embodiment, the biological sample of step (iii) is 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 certain embodiments, the biological sample of step (iii) is dispensed in an amount of 50 μL. The dispensed amount is the amount per reaction vessel, for example, when the reaction vessel is a 96 deep-well plate, 50 μL of the biological sample may be dispensed into each well.

[0281] In one embodiment, the biological sample of step (iii) is dispensed in an amount equal to that of the cell lysis composition dispensed in step (i).

[0282] In certain embodiments, 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).

[0283] In the case of directly mixing the biological sample with the cell lysis composition, the collected sample may be immersed in the cell lysis composition described above.

[0284]

[0285] Step (iv): Incubation of the mixture

[0286] In step (iv), the mixture is incubated at 50℃ or higher by the heating element to extract nucleic acids from the biological sample.

[0287] Step (iv) is controlled by a controller in the automated liquid handling system.

[0288] The incubation in step (iv) weakens the cells in the biological sample, thereby inducing an environment in which nucleic acids are easily released to the outside of the cells, and inducing the chemicals in the cell lysis composition to actively react with the cells to lyse the cells.

[0289] In one embodiment, the mixture may be incubated at about 55℃ to 65℃. For this, the heating element may be controlled by a controller such that its temperature increases to 95℃ or higher. The increase in the temperature of the heating element to 95℃ or higher can ensure that the temperature of the mixture within the reaction vessel is maintained at a temperature of 55℃ to 65℃.

[0290] In certain embodiments, the heating element is controlled by a controller such that its temperature increases to 100℃. In certain embodiments, the heating element is controlled by a controller such that its temperature increases to 105℃.

[0291] The incubation in step (iv) may be performed for a time sufficient to extract the nucleic acids. The incubation time may vary depending on various factors such as the type of the organism,e.g., a pathogen, from which the nucleic acid is to be extracted, the type of the biological sample, the components of the cell lysis composition, etc. One skilled in the art will be able to determine an appropriate incubation time, based on the correlation between the incubation time and the concentration of the extracted nucleic acid or between the incubation time and the efficiency or sensitivity of the nucleic acid amplification reaction.

[0292] In one embodiment, the incubation of step (iv) is carried out 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 therebetween. In certain embodiments, the incubation of step (iv) is carried out for about 10 minutes.

[0293]

[0294] The method of the present disclosure does not comprise purification of nucleic acids other than lysis of cells using the cell lysis composition described above. Specifically, the method of the present disclosure does not comprise purifying the nucleic acids, such as binding the nucleic acids to a solid support, washing, or eluting, after cell lysis.

[0295] The method of the present disclosure produce lysates of cells, which can be directly applied to nucleic acid amplification reactions.

[0296]

[0297] Hereinafter, the present invention will be described in more detail with reference to Examples. These Examples are provided only for illustration, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these Examples according to the gist of the present invention.

[0298]

[0299] EXAMPLES

[0300] Example 1: Determination of Optimal Concentration of Cell Lysis Components

[0301] To determine the optimal concentration of each of (i) guanidine hydrochloride (GuHCl), (ii) cetyltrimethylammonium bromide (CTAB), (iii) polyethylene glycol 200 (PEG200), (iv) n-lauroylsarcosine (NLS), and (v) polyoxyethylene sorbitan monolaurate (Tween 20) as cell lysis components, various concentrations of each of the above components were added to a nucleic acid amplification reagent to investigate their effect on the performance of PCR (Ct value, and RFU (relative fluorescence unit)).

[0302] First,Streptococcus pyogenes, a Group A Streptococcus (GAS) obtained from ZeptoMetrix, was spiked in a tube (prefilled with 1 mL of Liquid Amies transport medium) of ESwab® 480C (Copan Diagnostics Inc.) to a concentration of 6.02 X 106CFU / mL.

[0303] Then, various concentrations of the following five components were added as experimental groups, respectively: (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%.

[0304] Meanwhile, for comparison, TE buffer (Tris 10 mM and EDTA 1 mM, pH 8.0) as a control was added instead of the above components.

[0305] Thereafter, each of the experimental groups and the control were incubated at 25℃ for 10 minutes to lyse the cells ofS. pyogenes.

[0306] Then, a nucleic acid amplification reagent composed of primers and a probe for amplifying / detecting the target nucleic acid ofS. pyogenesand an enzyme mix for PCR (including Hot-startTaqpolymerase and dNTPs) was prepared. The primers and probe were designed to mimic the sequences under Centers for Disease Control and Prevention (CDC) Guidelines (Streptococcus Laboratory Resources and Protocols) with slight modification, which are shown in Table 1 below.

[0307] NameSequenceSEQ ID NOspy-F5'-GCACTCGCTACTATTTCTTACCTCAA-3'1spy-R5'-GTCACAATGTCTTGGAAACCAGTAAT-3'2spy-P5'-FAM-CCGCAACTCATCAAGGATTTCTGTTACCA-BHQ1-3'3

[0308] Thereafter, 5 μL of each of the experimental groups or the control and 15 μL of the nucleic acid amplification reagent were mixed to prepare a reaction mixture. The tube containing the reaction mixture was placed in the CFX96 Real-Time PCR thermal cycler (Bio-Rad), and subjected to 5 cycles of 5 sec at 95℃ and 20 sec at 60℃ and 40 cycles of 2 sec at 95℃ and 5 sec at 60℃ to amplify the target nucleic acid. The signal was measured at 60℃ per each cycle to obtain an amplification curve. Thereafter, a threshold value of RFU (relative fluorescence unit) 110 was applied to the amplification curve to calculate a Ct value, and RFU at the last cycle was recorded.

[0309]

[0310] <1-1> GuHCl

[0311] The results for the PCR reactions with different concentrations of GuHCl are shown in Table 2 below. Each result is an average of three replicates.

[0312] ReactionControlGuHCl30 mM100 mM300 mM500 mM1000 mMCt value#133.5933.3633.6537.31N / AN / A#232.8533.0433.4937.14N / AN / A#332.6932.8933.4837.52N / AN / AAverage33.0433.1033.5437.32--Experimental group - Control-0.050.504.28--Standard Deviation0.480.240.100.19--RFU#11306.271372.051459.66997.4911.923.88#21496.991527.311451.131198.619.541.53#31395.811487.471434.551034.5710.157.17Average1399.691462.281448.451076.8910.544.19Experimental group - Control-62.5948.76-322.80-1389.15-1395.50

[0313] As shown in Table 2, when GuHCl was added in an amount of 500 mM or more, it was found that the Ct value was not calculated and the RFU value was also significantly low, indicating the inhibitory effect of 500 mM or more of GuHCl on the PCR reaction.

[0314] The results demonstrate that it is preferred to add GuHCl in an amount of about 30 to about 300 mM.

[0315]

[0316] <1-2> CTAB

[0317] The results of the PCR reactions with different concentrations of CTAB are shown in Table 3 below. Each result is an average of three replicates.

[0318] ReactionControlCTAB0.05 wt%0.1 wt%0.5 wt%1.0 wt%1.5 wt%Ct value#131.7227.2528.42N / AN / AN / A#231.4027.3428.44N / AN / AN / A#331.4427.2527.38N / AN / AN / AAverage31.5227.2828.08---Experimental group - Control--4.24-3.44---Standard Deviation0.170.050.61---RFU#123352900261214-9-15#224272906264822-3-8#325832820278018-3-7Average24482875268018-5-10Experimental group - Control-427.00231.67-2430.20-2453.15-2458.25

[0319] As shown in Table 3, when 0.5 wt%, 1.0 wt%, and 1.5 wt% of CTAB were added, the Ct value was not calculated and the RFU value was also significantly low, indicating the inhibitory effect of 0.5 wt% or more of CTAB on the PCR reaction.

[0320] The results demonstrate that it is preferred to add CTAB in an amount of about 0.05 wt% to less than 0.5 wt%.

[0321]

[0322] <1-3> PEG200

[0323] The results of the PCR reactions with different concentrations of PEG200 are shown in Tables 4 and 5 below. Each result is an average of three replicates.

[0324] ReactionControlPEG2005 wt%10 wt%15 wt%20 wt%Ct Value#133.3532.9232.2932.6031.84#232.2732.5632.1432.1232.25#332.5432.8632.3032.2532.32Average32.7232.7832.2432.3232.14Experimental group - Control-0.06-0.48-0.40-0.58Standard Deviation0.560.190.090.250.26RFU#11180.201307.151423.391264.361318.41#21192.161413.181385.731419.791270.66#31199.841309.071375.361320.241266.19Average1190.731343.131394.821334.801285.09Experimental group - Control-152.40204.09144.0694.36

[0325] ReactionControlPEG20030 wt%40 wt%50 wt%60 wt%70 wt%80 wt%Ct value#131.7230.8430.9031.1629.2429.5629.18#231.4030.7731.4330.7529.5330.0929.95#331.4430.8531.4930.7429.9928.8828.76Average31.5230.8231.2730.8829.5829.5129.30Experimental group - Control--0.70-0.25-0.64-1.94-2.01-2.22Standard Deviation0.170.040.330.240.380.610.43RFU#12335233523842097232821372103#22427245723242308230120412067#32583235922262241230323772331Average2448238423112215231121852167Experimental group - Control--64.67-137.00-233.00-137.67-263.33-280.62

[0326] As shown in Tables 4 and 5, when PEG200 was added, the Ct value was decreased, but the RFU value was slightly decreased. However, it was found that PEG200, even when added in large amounts, had no significant effect on the PCR reaction.

[0327] The results demonstrate that 5 wt% to 80 wt% of PEG200 has no significant effect on the PCR reaction.

[0328]

[0329] <1-4> NLS

[0330] The results of the PCR reaction with different concentrations of NLS are shown in Table 6 below. Each result is an average of three replicates.

[0331] ReactionControlNLS0.05 wt%0.1 wt%0.3 wt%0.5 wt%1 wt%1.5 wt%Ct value#133.3532.8633.4532.9134.33N / AN / A#232.2731.9633.2432.7434.21N / AN / A#332.5432.1432.9533.0134.00N / AN / AAverage32.7232.3233.2132.8934.18--Experimental group - Control--0.400.490.171.46--Standard Deviation0.560.170.250.140.17--RFU#11180.201386.491321.441428.901342.391.571.58#21192.161441.691447.401390.891410.091.971.41#31199.841366.721379.191371.271388.295.142.51Average1190.731405.311382.671397.021380.252.891.83Experimental group - Control209.32152.40191.94206.29189.52-1187.84-1188.90

[0332] As shown in Table 6, the addition of 1 wt% and 1.5 wt% of NLS did not produce a Ct value and showed a significant decrease in the RFU value, indicating the inhibitory effect of 1 wt% to 1.5 wt% of NLS on the PCR reaction.

[0333] The results demonstrate that it is preferred to add NLS in an amount of about 0.05 wt% to less than 1 wt%.

[0334]

[0335] <1-5> Tween 20

[0336] The results of the PCR reactions with different concentrations of Tween 20 are shown in Table 7 below. Each result is an average of three replicates.

[0337] ReactionControlTween 205 wt%10 wt%15 wt%20 wt%25 wt%Ct value#133.3531.1931.4431.4531.4131.10#232.2731.1931.1630.8331.2830.78#332.5431.7531.4531.1231.2731.15Average32.7231.3831.3531.1331.3231.01Experimental group - Control--1.34-1.37-1.59-1.40-1.71Standard Deviation0.560.320.160.310.080.20RFU#11180.201156.601045.551074.70912.31914.67#21192.161131.861146.891032.42998.57949.03#31199.841175.171063.07983.14947.20909.12Average1190.731154.541085.171030.09952.69924.27Experimental group - Control0.00-36.19-105.56-160.65-238.04-266.46

[0338] As shown in Table 7, when Tween 20 was added in an amount of 20 wt% or 25 wt%, the RFU values were slightly decreased.

[0339] The results demonstrate that it is preferred to add Tween 20 in an amount of about 5 wt% to 15 wt%.

[0340]

[0341] Example 2: Effect of pH of Cell Lysis Composition

[0342] In order to examine the effect of pH of the cell lysis composition according to the present disclosure, cell lysis compositions having the same components but different pH values were prepared.

[0343] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0344] Then, as experimental groups, 10 mM of Tris, 1 mM of EDTA, 100 mM of GuHCl, 0.05 wt% of NLS, 5 wt% of Tween 20, 15 wt% of PEG8000, and 5 wt% of 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 each of the mixtures was added to the tube and incubated at 25℃ for 10 minutes to lyse the cells ofS. pyogenes.

[0345] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0346] Then, the experimental group or the control was subjected to PCR using the nucleic acid amplification reagent as in Example 1, thereby obtaining an amplification curve, and the Ct value was calculated from the amplification curve.

[0347] The results are shown in Table 8 below. Each result is an average of three replicates.

[0348] ControlEx. grouppH 8Ex. grouppH 9Ex. grouppH 10Ex. grouppH 11Ex. grouppH 12#131.5131.3631.0530.7732.9931.17#231.3831.3031.4530.5232.1832.09#331.0631.1931.9432.0031.6131.73Average31.3231.2831.4831.0932.2631.66Experimental group - Control--0.030.16-0.220.940.35Standard Deviation0.230.090.440.790.700.46

[0349] As shown in Table 8, 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 between 8 and 9, it was deemed desirable to adjust the pH of the composition to 8 as well.

[0350]

[0351] Example 3: Effect of CTAB and PEG combination

[0352] To examine the effect of the combination of CTAB and PEG on cell lysis, compositions comprising the combination of CTAB and PEG were compared with other compositions.

[0353] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0354] Then, (i) a composition containing GuHCl and PEG (Experimental Group 1), (ii) a composition containing a low concentration of CTAB and PEG (Experimental Group 2), (iii) a composition containing a high concentration of CTAB and PEG (Experimental Group 3), and (iv) a composition containing only PEG without GuHCl and CTAB (Experimental Group 4) were prepared as experimental groups. Then, 100 μL of each of the compositions was added to the tube and incubated at 25℃ for 10 minutes to lyse the cells ofS. pyogenes.

[0355] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0356] Then, the experimental group or the control was subjected to PCR using a nucleic acid amplification reagent as in Example 1, thereby obtaining an amplification curve, and the Ct value was calculated from the amplification curve.

[0357] The results are shown in Table 9 below. Each result is an average of three replicates.

[0358] ReactionControlEx. group 1Ex. group 2Ex. group 3Ex. group 4Composition of reagentTris10 mM10 mM10 mM10 mMEDTA1 mM1 mM1 mM1 mMGuHCl100 mM---CTAB-0.1 wt%0.5 wt%-NLS0.05 wt%0.05 wt%0.05 wt%-Tween 205 wt%5 wt%5 wt%5 wt%PEG20060 wt%--60 wt%PEG8000-15 wt%15 wt%-pH8888#131.4430.2628.4028.6931.19#231.2029.9228.5528.4531.49#331.0129.7528.4428.7232.11Average31.2229.9828.4628.6231.60Experimental group - Control--1.24-2.76-2.600.38Standard Deviation0.220.260.080.150.47

[0359] As shown in Table 9, 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, indicating excellent PCR reactions of experimental groups 2 and 3 containing CTAB and PEG.

[0360] The results demonstrate that using a cell lysis composition comprising a combination of CTAB and PEG can improve the detection performance of the target nucleic acid.

[0361]

[0362] Example 4: Effects of GuHCl

[0363] In order to examine the effect of GuHCl on cell lysis, compositions containing GuHCl was compared with compositions not containing GuHCl.

[0364] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0365] Then, (i) a composition containing no GuHCl but containing a low concentration of CTAB, and PEG (Experimental Group 1), (ii) a composition containing no GuHCl but containing a high concentration of CTAB, and PEG (Experimental Group 2), (iii) a composition containing a low concentration of GuHCl, a low concentration of CTAB, and PEG (Experimental Group 3), and (iv) a composition containing a high concentration of GuHCl, a low concentration of CTAB, and PEG (Experimental Group 4) were prepared as experimental groups. Then, 100 μL of each of the compositions was added to the tube and incubated at 25℃ for 10 minutes to lyse the cells ofS. pyogenes.

[0366] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0367] Then, the experimental group or the control was subjected to PCR using a nucleic acid amplification reagent as in Example 1, thereby obtaining an amplification curve, and the Ct value and the RFU at the last cycle was recorded.

[0368] The results are shown in Table 10 below. Each result is an average of three replicates.

[0369] ReactionControlEx. group 1Ex. group 2Ex. group 3Ex. group 4Composition of reagentTris10 mM10 mM10 mM10 mMEDTA1 mM1 mM1 mM1 mMGuHCl--100 mM250 mMCTAB0.1 wt%0.5 wt%0.1 wt%0.1 wt%NLS0.05 t%0.05 wt%0.05 t%0.05 wt%Tween 205 wt%5 wt%5 wt%5 wt%PEG800015 wt%15 wt%15 wt%15 wt%pH8888Ct value#130.5028.8829.0629.1930.04#231.2029.0328.4929.0229.87#331.2928.6828.4329.0230.08Average31.0028.8728.6629.0830.00Experimental group - Control--2.13-2.34-1.92-1.00Standard Deviation0.430.180.340.100.11RFU#11340.141066.46869.791154.411258.12#21343.511069.89927.941166.181212.80#31328.771073.35877.351153.351191.89Average1337.481069.90891.701157.981220.94Experimental group - Control--267.57-445.78-179.50-116.54Standard Deviation1340.141066.46869.791154.411258.12

[0370] As shown in Table 10, experimental groups 1 and 2 containing no GuHCl but containing CTAB and PEG showed decreased Ct values but decreased RFU values. In contrast, experimental groups 3 and 4 each containing GuHCl, CTAB, and PEG showed improved RFU values compared to experimental groups 1 and 2 due to the addition of GuHCl.

[0371] The results demonstrate that the decreased in RFU values due to the use of CTAB and PEG can be improved by the addition of GuHCl.

[0372]

[0373] Example 5: Effects of PEG200

[0374] In order to examine the effect of the molecular weight of PEG on cell lysis, compositions containing PEG200 or PEG8000 were compared.

[0375] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0376] Then, (i) a composition containing PEG8000 (Experimental group 1), and (ii) a composition containing PEG200 (Experimental group 2) were prepared as experimental groups.

[0377] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0378] Then, the experimental group or the control was subjected to PCR using a nucleic acid amplification reagent as in Example 1, thereby obtaining an amplification curve, and the Ct value and the RFU at the last cycle was recorded.

[0379] The results are shown in Table 11 below. Each result is an average of three replicates.

[0380] ReactionControlEx. group 1Ex. group 2Composition of reagentTris10 mM10 mMEDTA1 mM1 mMGuHCl100 mM100 mMCTAB0.1 wt%0.1 wt%NLS0.05 wt%0.05 wt%Tween 205 wt%5 wt%PEG200-60 wt%PEG800015 wt%-pH88Ct value#131.3729.5829.38#231.0229.2529.58#330.7229.0529.55Average31.0429.2928.50Experimental group - Control--1.74-2.53Standard Deviation0.330.270.12RFU#11268.871094.751013.93#21325.631092.261030.55#31334.751112.63983.36Average1309.751099.881009.28Experimental group - Control--209.87-300.47

[0381] As shown in Table 11, experimental group 2 containing PEG200 at a concentration of 60 wt% exhibited excellent amplification performance of the target nucleic acid (decreased Ct value), compared to experimental group 1 containing PEG8000 at a concentration of 15 wt%.

[0382] In addition, the solution of experimental group 1 containing PEG8000 (powder state) at a concentration of 15 wt% showed poor tractability due to many bubbles and high viscosity, whereas experimental group containing PEG200 (liquid state) showed good tractability.

[0383]

[0384] Example 6: Effect of Tris and PEG200

[0385] In order to examine the effect of Tris and PEG200 on cell lysis, compositions containing different concentrations of Tris and PEG200 were compared.

[0386] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0387] Then, (i) a composition containing 10 mM of Tris and 60 wt% of PEG200 (Experimental Group 1), (ii) a composition containing 50 mM of Tris and 60 wt% of PEG200 (Experimental Group 2), and (iii) a composition containing 10 mM of Tris (Experimental Group 3) were prepared as experimental groups.

[0388] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0389] Then, the experimental group or the control was subjected to PCR using a nucleic acid amplification reagent as in Example 1, thereby obtaining an amplification curve, and the Ct value and the RFU at the last cycle was recorded.

[0390] The results are shown in Table 12 below. Each result is an average of three replicates.

[0391] ReactionControlEx. group 1Ex. group 2Ex. group 3Composition of reagentTris105010EDTA111GuHCl100100100CTAB0.10.10.1NLS0.050.050.05Tween 20555PEG2006060-pH888Ct value#133.0230.7631.1032.96#232.8830.7631.2632.94#332.8830.7230.7032.90Average32.9330.7531.0232.93Experimental group - Control--2.14-1.860.05Standard Deviation0.080.020.290.03RFU#12181.171887.891999.241607.10#22260.741882.382181.531847.46#32262.681919.092068.281538.23Average2234.861896.452083.021664.26Experimental group - Control--366.23-179.66-598.42

[0392] As shown in Table 12, experimental group 1 containing 10 mM of Tris exhibited a Ct value which was approximately 0.3 lower than that of experimental group 2 containing 50 mM of Tris. This indicates that using as little as 10 mM of Tris is advantageous for the detection of target nucleic acids.

[0393] Meanwhile, it was found that experimental groups 1 and 2 containing PEG200 had lower Ct values and higher RFU values compared to experimental group 3 containing no PEG200. The results demonstrate that addition of PEG200 is beneficial for cell lysis.

[0394]

[0395] Example 7: Analytical Sensitivity of Cell lysis Composition

[0396] In order to investigate the analytical sensitivity of PCR using the cell lysis composition according to the present disclosure, target nucleic acids were obtained from three strains which are causative agents of pharyngitis,Streptococcus pyogenes,Streptococcus dysgalactiae subsp. Equisimilis, andArcanobacterium haemolyticum, using the cell lysis composition, and then immediately subjected to PCR.

[0397] Specifically, each of 5-fold serial dilutions,i.e., 6.02 X 106CFU / mL to 6.02 X 102CFU / mL ofStreptococcus pyogenes, 5-fold serial dilutions,i.e., 4.14 X 104CFU / mL to 4.14 X 100CFU / mL ofStreptococcus dysgalactiae subsp. Equisimilis, and 5-fold serial dilutions,i.e., 6.12 X 104CFU / mL to 6.12 X 101CFU / mL ofArcanobacterium haemolyticum, all obtained from ZeptoMetrix Inc., was spiked in the first to fifth tubes of ESwab® 480C (Copan Diagnostics Inc.), respectively.

[0398] Thereafter, 100 μL of the sample and 100 μL of the cell lysis composition according to the present disclosure (10 mM of Tris, 1 mM of EDTA, 100 mM of GuHCl, 0.1 wt% of CTAB, 0.05 wt% of NLS, 5 wt% of Tween 20, and 60 wt% of PEG200; pH 8) as an experimental group were added, and then incubated at 25℃ for 10 minutes to lyse cells of each strain.

[0399] Meanwhile, as a control for comparison, a commercially available extraction reagent (STARMag 96x4 Universal Cartridge kit, 744300.4, Seegene Inc.) was added to the tube to extract a target nucleic acid.

[0400] Thereafter, a nucleic acid amplification reagent comprising primers and a probe for amplifying and detecting a target nucleic acid of each strain and an enzyme mix for PCR (including Hot-startTaqpolymerase and dNTPs) was prepared. Sequences of the primers and the probe are shown in Table 13 below.

[0401] NameSequenceSEQ IDspy-F5'-GCACTCGCTACTATTTCTTACCTCAA-3'1spy-R5'-GTCACAATGTCTTGGAAACCAGTAAT-3'2spy-P5'-FAM-CCGCAACTCATCAAGGATTTCTGTTACCA-BHQ1-3'3SDSE-F5'-CGCGAATCATTGCTCTGTGG-3'4SDSE-R5'-AACCGATGGTGTGGAAAGCA-3'5SDSE-P5'-HEX-ACCGCAACCATTTCTCGATTTGCT-BHQ1-3'6AH-F5'-CGCGAACGCTCTGGAAATT-3'7AH-R5'-GCCGTATCGCCAGCACTAGT-3'8AH-P5'-C610-ATTTTACTGCGTGGCGCC-BHQ1-3'9

[0402] Thereafter, 5 μL of the experimental group or the control and 15 μL of the nucleic acid amplification reagent were mixed to prepare a reaction mixture. The tube containing the reaction mixture was placed in the CFX96 Real-Time PCR thermal cycler (Bio-Rad), and subjected to 5 cycles of 5 sec at 95℃ and 20 sec at 60℃ and 40 cycles of 2 sec at 95℃ and 5 sec at 60℃ to amplify the target nucleic acid. The signal was measured at 60℃ per each cycle to obtain an amplification curve. Thereafter, a threshold value of RFU (relative fluorescence unit) 110 was applied to the amplification curve to calculate a Ct value.

[0403] The results are shown in Table 14 below. Each result is an average of three replicates.

[0404] StrainConcentrationControlExperimental groupCt valueAverageCt valueAverageStreptococcus pyogenes6.02 X 106CFU / mL29.5029.5027.6827.7929.5427.5129.4728.176.02 X 105CFU / mL32.7232.9431.1931.2832.8330.9433.2831.726.02 X 104CFU / mL35.8735.8635.0634.9434.9934.3836.7335.396.02 X 103CFU / mLN / A-37.9738.95N / A41.34N / A37.536.02 X 102CFU / mLN / A-44.62-

[0405] StrainConcentrationControlExperimental groupCt valueAverageCt valueAverageStreptococcus dysgalactiae subsp. Equisimilis4.14 X 104CFU / mL31.9331.9526.9527.6731.9928.0131.9428.064.14 X 103CFU / mL35.7436.0130.7730.8537.1131.0935.1630.694.14 X 102CFU / mLN / A-34.0634.21N / A34.39N / A34.184.14 X 101CFU / mLN / A-38.78-N / A35.36N / AN / A4.14 X 100CFU / mLN / A-N / A-

[0406] StrainConcentrationControlExperimental groupCt valueAverageCt valueAverageArcanobacterium haemolyticum6.12 X 105CFU / mL26.9826.9026.1725.9826.9025.8126.8225.966.12 X 104CFU / mL30.7930.6629.3029.3730.7529.5030.4429.306.12 X 103CFU / mL34.6433.9932.9634.4733.7034.9433.6335.526.12 X 102CFU / mL37.15-N / A-36.0535.98N / A34.226.12 X 101CFU / mLN / A-N / A-

[0407] As shown in Table 14, the control was able to detectStreptococcus pyogenesat concentrations of 6.02 X 106CFU / mL to 6.02 X 104CFU / mL, whereas the experimental group was able to detectStreptococcus pyogenesat concentrations of 6.02 X 106CFU / mL to 6.02 X 103CFU / mL. The results demonstrate that the PCR reaction using the cell lysis composition according to the present disclosure has a higher analytical sensitivity in detectingStreptococcus pyogenesthan the PCR reaction using the conventional extraction method.

[0408] As shown in Table 15, the control was able to detectStreptococcus dysgalactiae subsp. Equisimilisat concentrations of 4.14 X 104CFU / mL to 4.14 X 103CFU / mL, while the experimental group was able to detectStreptococcus dysgalactiae subsp. Equisimilisat concentrations of 4.14 X 104CFU / mL to 4.14 X 102CFU / mL. The results demonstrate that the PCR reaction using the cell lysis composition according to the present disclosure has a higher analytical sensitivity in detectingStreptococcus dysgalactiae subsp. Equisimilisthan the PCR reaction using the conventional extraction method.

[0409] As shown in Table 16, the control was able to detectArcanobacterium haemolyticumat concentrations of 6.12 X 105CFU / mL to 6.12 X 103CFU / mL, and the experimental group was also able to detectArcanobacterium haemolyticumat concentrations of 6.12 X 105CFU / mL to 6.12 X 103CFU / mL. The results demonstrate that the PCR reaction using the cell lysis composition according to the present disclosure has the analytical sensitivity in detectingArcanobacterium haemolyticumequivalent to the PCR reaction using the conventional extraction method.

[0410] Taken together, the PCR reaction using the cell lysis composition according to the present disclosure has an excellent or equivalent analytical sensitivity to the PCR reaction using the conventional nucleic acid extraction, which demonstrates that the cell lysis composition according to the present disclosure enables a simple and rapid PCR reaction without purifying nucleic acids.

[0411]

[0412] Example 8: Effect of Pre-incubation of Cell Lysis Compositions in Automated Liquid Handling System

[0413] In order to examine the effect of the nucleic acid extraction method according to the present disclosure comprising pre-incubation of a cell lysis composition in an automated liquid handling system, nucleic acids were extracted by the method according to one embodiment of the present disclosure from three strains that are causative agents of pharyngitis, namelyStreptococcus pyogenes,Streptococcus dysgalactiae subsp. Equisimilis, andArcanobacterium haemolyticum, and then immediately subjected to PCR reactions.

[0414] Specifically, tubes each containing a cell lysis composition and tubes each containing a biological sample were mounted on an automated liquid handling system (SEEGENE STARlet, dimension: 1124 (W) x 795 (D) x 903 (H) mm).

[0415] The cell lysis composition was composed of 10 mM of Tris, 1 mM of EDTA, 100 mM of GuHCl, 0.1 wt% of CTAB, 0.05 wt% of NLS, 5 wt% of Tween 20, and 60 wt% of PEG200; pH 8.

[0416] The biological sample was prepared by spiking each of 6.02X106CFU / ml ofStreptococcus pyogenesobtained from ZeptoMetrix Inc., 4.14X104CFU / mL ofStreptococcus dysgalactiae subsp. equisimilis, and 6.12X105CFU / mL ofArcanobacterium hamolyticum, in first to third tubes of ESwab® 480C (Copan Diagnostics Inc.), and then transferring a portion of the spiked solution to new tubes.

[0417] Afterwards, an empty 96 deep-well plate was mounted on a Hamilton heater shaker in the automated liquid handling system.

[0418] The empty 96 deep-well plate was then heated by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the automated liquid handling system.

[0419] 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.

[0420] The cell lysis composition was then heated for 10 minutes by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the automated liquid handling system. During the heating, the maximum temperature of the cell lysis composition was determined. It was found that the temperature of the cell lysis composition was maintained at about 58-60℃.

[0421] Next, 50 μL of the biological sample was dispensed into each well of the 96 deep-well plate containing the cell lysis composition by the controller of the automated liquid handling system. The biological sample and the cell lysis composition were mixed thoroughly by pipetting up and down using a pipetting channel.

[0422] The mixture was then heated for 10 minutes by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the automated liquid handling system, thereby obtaining cell lysates.

[0423]

[0424] Thereafter, a nucleic acid amplification reagent composed of primers and probes for amplifying / detecting target nucleic acids of each strain and an enzyme mix for PCR (including Hot-startTaqpolymerase and dNTPs) was prepared. The sequences of the primers and probes are as in Table 13 of Example 7.

[0425] Thereafter, 5 μL of the cell lysate and 15 μL of the nucleic acid amplification reagent were mixed to prepare a reaction mixture. The tube containing the reaction mixture was placed in the CFX96 Real-Time PCR thermal cycler (Bio-Rad), and subjected to 5 cycles of 5 sec at 95℃ and 20 sec at 60℃ and 40 cycles of 2 sec at 95℃ and 5 sec at 60℃ to amplify the target nucleic acid. The signal was measured at 60℃ per each cycle to obtain an amplification curve. Thereafter, a threshold value of RFU (relative fluorescence unit) 110 was applied to the amplification curve to calculate a Ct value.

[0426] The results are shown in Table 17. Each result is an average of three replicates.

[0427] StrainCt valueStreptococcus pyogenes27.87Streptococcus dysgalactiae subsp. equisimilis29.69Arcanobacterium haemolyticum26.10

[0428] As can be seen in Table 17, it was found that the cell lysates obtained by the method of the present disclosure comprising pre-incubating the cell lysis composition provided excellent detection results without inhibition against the nucleic acid amplification reaction.

[0429]

[0430] Comparative Example 9: Effect of Pre-incubation of Biological Samples in Automated Liquid Handling System

[0431] In order to compare the effect of pre-incubation of the cell lysis composition on nucleic acid extraction with that of pre-incubation of the biological sample, the biological samples were pre-incubated (the cell lysis composition was not pre-incubated) to extract nucleic acids, which were subjected to a nucleic acid amplification reaction.

[0432] Specifically, an empty 96 deep-well plate was heated by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the same automated liquid handling system used in Example 8.

[0433] 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.

[0434] The biological sample was then heated for 10 minutes by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the automated liquid handling system. During the heating, the maximum temperature of the biological sample was determined. It was found that the temperature of the biological sample was maintained at about 58-60℃.

[0435] Next, 50 μL of the cel lysis composition was dispensed into each well of the 96 deep-well plate containing the biological sample by the controller of the automated liquid handling system. The biological sample and the cell lysis composition were mixed thoroughly by pipetting up and down using a pipetting channel.

[0436] The mixture was then heated for 10 minutes by increasing the temperature of the Hamilton heater shaker to a predefined temperature, 100℃ by the controller of the automated liquid handling system, thereby obtaining cell lysates.

[0437]

[0438] Then, real-time PCR was performed in the same manner as in Example 8, and a Ct value was calculated.

[0439] The results are shown in Table 18. Each result is an average of three replicates.

[0440] StrainCt valueExample 8 - Comparative Example 9Streptococcus pyogenes29.29- 1.42Streptococcus dysgalactiae subsp. equisimilis32.00- 2.31Arcanobacterium haemolyticum27.64- 1.54

[0441] As shown in Table 18, it was found that the nucleic acid extraction method (Comparative Example 9) comprising pre-incubating the biological sample showed higher Ct values compared to the nucleic acid extraction method (Example 8) comprising pre-incubating the cell lysis composition. This demonstrates that pre-incubating the biological sample has a lower efficiency of the nucleic acid amplification reaction compared to pre-incubating the cell lysis composition.

[0442]

[0443] Example 10: Effect of Pre-incubation Time of Cell Lysis Composition in Automated Liquid Handling System

[0444] In order to examine the effect of the pre-incubation time of the cell lysis composition on the nucleic acid amplification reaction in the automated liquid handling system, the cell lysis composition was pre-incubated for different times, and the obtained nucleic acids were subjected to a real-time PCR reaction as in Example 8.

[0445] Specifically, the process described in Example 8 was repeated, except that the three strains were spiked in the following quantities.

[0446] -Streptococcus pyogenes(ZeptoMetrix): 6.02X105CFU / mL, 6.02X104CFU / mL, and 6.02X103CFU / mL;

[0447] -Streptococcus dysgalactiae subsp. Equisimilis(ATCC): 1.4X105CFU / mL, 1.4X104CFU / mL, and 1.4X103CFU / mL;

[0448] -Arcanobacterium hamolyticum(ATCC): 1X104CFU / mL, 1X103CFU / mL, and 1X102CFU / mL.

[0449] The results ofStreptococcus pyogenes,Streptococcus dysgalactiae subsp. equisimilis, andArcanobacterium hamolyticumare shown in Tables 19, 20, and 21, respectively. Each result is an average of three replicates.

[0450] QuantityEx. Group 1(pre-heatingfor 5 minutes)Ex. Group 2(Pre-heatingfor 10 minutes)Ex. Group 3(Pre-heatingfor 15 minutes)Ex. Group 4(Pre-heatingfor 20 minutes)Ct valueAverageCt valueAverageCt valueAverageCt valueAverage6.02 X 105CFU / mL31.2931.1430.9827.7930.9030.5430.4930.6031.2730.4830.8130.6130.8530.9629.9030.716.02 X 104CFU / mL33.3133.4433.5933.4433.8333.9132.9433.4433.0633.2733.0933.8533.9633.4534.8233.546.02 X 103CFU / mL37.3437.3136.7737.9039.1940.8335.8036.4138.4739.5338.4937.4336.1237.3944.8035.99

[0451] QuantityEx. Group 1(pre-heatingfor 5 minutes)Ex. Group 2(Pre-heatingfor 10 minutes)Ex. Group 3(Pre-heatingfor 15 minutes)Ex. Group 4(Pre-heatingfor 20 minutes)Ct valueAverageCt valueAverageCt valueAverageCt valueAverage1.4 X 105CFU / mL33.7633.4432.8727.7933.3232.6032.7232.7833.4932.4432.5532.7133.0832.5631.9332.901.4 X 104CFU / mL36.4436.0535.1833.4435.3835.4835.1735.6735.6236.2235.0536.0136.1035.5236.0335.811.4 X 103CFU / mLN / A-41.81-39.1438.45N / A-43.44N / A38.18N / AN / A38.0338.0240.73

[0452] QuantityEx. Group 1(pre-heatingfor 5 minutes)Ex. Group 2(Pre-heatingfor 10 minutes)Ex. Group 3(Pre-heatingfor 15 minutes)Ex. Group 4(Pre-heatingfor 20 minutes)Ct valueAverageCt valueAverageCt valueAverageCt valueAverage1 X 104CFU / mL30.2029.5729.4529.5730.0629.5629.3229.6529.0229.3429.5729.9528.6329.9129.0429.691 X 103CFU / mL31.5332.3534.1332.5831.8732.2932.8633.2133.1131.2531.8233.1332.4032.3733.1633.641 X 102CFU / mL38.16-37.9537.2937.50-39.51-N / A37.75N / AN / AN / A36.15N / A34.60

[0453] As shown in Table 19, in the case ofStreptococcus Pyogenes, Experimental Group 2 pre-heated for 10 minutes showed overall low Ct values.

[0454] In addition, as shown in Table 20, in the case ofStreptococcus dysgalactiae subsp. equisimilis, Experimental Group 2 pre-heated for 10 minutes showed overall low Ct values, and Experimental Group 3 and Experimental Group 2 were able to detect small quantities of strains.

[0455] Further, as shown in Table 21, in the case ofArcanobacterium haemolyticum, all Experimental Groups showed similar Ct values, and Experimental Group 2 was able to detect a low quantity of strains.

[0456] As described above, it was found that the optimal pre-heating time of the cell lysis composition is 10 minutes, in terms of efficiency, sensitivity, and TAT of the nucleic acid amplification reaction.

[0457] Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

A method for extracting nucleic acids from a biological sample in an automated liquid handling system, comprising:(i) dispensing a cell lysis composition into an empty reaction vessel, mounted on a heating element in the automated liquid handling system, wherein the cell lysis composition serves to lyse cells in the biological sample;(ii) pre-incubating the cell lysis composition at 50℃ 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℃ or higher by the heating element to extract nucleic acids from the biological sample;wherein steps (i)-(iv) are controlled by a controller and steps (i) and (iii) are performed by means of pipetting channels in the automated liquid handling system.The method of claim 1, wherein the biological sample is a swab, saliva, or a mixture thereof from a subject.The method of claim 1, wherein the biological sample contains or is suspected of containingStreptococcus pyogenes,Streptococcus equi subsp. Equi,Streptococcus equi subsp. zooepidemicus,Streptococcus dysgalactiae subsp. dysgalactiae,Streptococcus dysgalactiae subsp. equisimilis,Streptococcus canis,Arcanobacterium haemolyticum, or combinations thereof.The method of claim 1, wherein the heating element serves to heat the reaction vessel mounted thereon to a predefined temperature while shaking.The method of claim 1, wherein the reaction vessel is a deep-well plate or a well plate.The method of 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.The method of claim 1, wherein the cell lysis composition comprises GuHCl in an amount of 30 to 300 mM, CTAB in an amount of 0.05 to less than 0.5 wt% based on the total weight of the cell lysis composition, and PEG in an amount of 5 to 80 wt% based on the total weight of the cell lysis composition.The method of claim 6, wherein the cell lysis composition further comprises a detergent, a chelating agent, a buffering agent, or combinations thereof.The method of claim 1, wherein the cell lysis composition of step (i) is dispensed in an amount of 10 to 100 μL.The method of claim 1, the method further comprises heating the empty reaction vessel by the heating element in the automated liquid handling system, prior to step (i).The method of claim 10, wherein the heating element of step (ii) is controlled by the controller such that its temperature increases to 95℃ or higher.The method of claim 1, wherein the heating element of step (ii) is controlled by the controller such that its temperature remains at 95℃ or higher.The method of claim 1, wherein the pre-incubation of step (ii) is carried out for at least 5 minutes.The method of claim 1, wherein the biological sample of step (iii) is dispensed in an amount of 10 to 100 μL.The method of claim 1, wherein the biological sample of step (iii) is dispensed in an amount equal to that of the cell lysis composition dispensed in step (i).The method of claim 1, wherein the heating element of step (iv) is controlled by the controller such that its temperature increases to 95℃ or higher.The method of claim 1, wherein the extracted nucleic acid is directly applied to a nucleic acid amplification reaction without further purification.The method of claim 17, wherein the nucleic acid amplification reaction is PCR, real-time PCR, or Loop Mediated Isothermal Amplification (LAMP).