Composition for lysing cells in biological sample and method for detecting target nucleic acid using same

Abstract

The present invention relates to a composition for lysing cells in a biological sample, and a method for detecting target nucleic acids using the same. The composition of the present invention includes guanidine hydrochloride (GuHCl), cetyltrimethylammonium bromide (CTAB), and polyethylene glycol (PEG). The composition allows cell lysates to be directly used in a target nucleic acid amplification reaction without purification of the target nucleic acids, thereby reducing the time and cost required for the target nucleic acid amplification reaction.

Description

COMPOSITION FOR LYSING CELLS IN BIOLOGICAL SAMPLE AND METHOD FOR DETECTING TARGET NUCLEIC ACID USING SAME

[0001] Cross-Reference To Related Applications

[0002] This application claims priority from Korean Patent Application No. 10-2023-0142322, filed on October 23, 2023, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Technical Field

[0004] The present invention relates to a composition for lysing cells in a biological sample, and a method for detecting target nucleic acid using the same.

[0005]

[0006] Since its initial identification in 2019, SARS-CoV-2, which has caused a worldwide pandemic, has dramatically increased the demand for diagnostic kits capable of detecting infection of pathogens from biological samples in subjects.

[0007] In order to prevent the spread of a disease, a nucleic acid amplification method with high accuracy, in particular, real-time PCR, has been widely used. In addition, research is being actively conducted to reduce the time required for each step of the nucleic acid amplification method for rapid diagnosis.

[0008] For nucleic acid amplification, it is required to extract a target nucleic acid from a pathogen, such as a virus or bacteria, in a biological sample. Conventionally, extraction of target nucleic acids involves the steps of lysis, binding, washing, purification, and elution. Specifically, a biological sample is incubated with a lysis buffer to destroy a cell wall, a cell membrane, and the like and expose a nucleic acid; the exposed nucleic acid is bound to a specific structure such as a membrane, a bead, or the like; the nucleic acid is washed several times using buffers, and then the nucleic acid is separated from the structure to obtain a purified nucleic acid.

[0009] However, the above-described conventional nucleic acid extraction method is inefficient in terms of time and cost, because it requires equipment, reagents, and tools for each step, and its procedure is somewhat complicated. These drawbacks make the method unsuitable for situations requiring urgent or large-scale diagnosis.

[0010] In order to improve this, a method of obtaining nucleic acids by simply and rapidly lysing cells without purifying the nucleic acids, and a cell lysis composition used therein have been developed.

[0011] As an example, U.S. Patent Publication No. 2021-0363567 discloses a method of mixing a biological sample with a transport solution including a chaotropic agent and a chelating agent to obtain a stabilized sample, mixing the stabilized sample with an activating solution having a pH of 6.8 to 8.2, and then performing PCR. As another example, U.S. Patent Publication No. 2021-0115498 discloses a cell lysis composition comprising a chaotrope, a detergent, a chelator, a reducing agent, nuclease-free water, and a buffer, and a method for determining the presence of a target nucleic acid using the same.

[0012] However, since the above-described solution or composition contains components that inhibit amplification of a target nucleic acid or has a low cell lysis activity, amplification of a target nucleic acid using the solution or composition has a problem in that it shows a lower analytical sensitivity than that using conventional nucleic acid extraction.

[0013] Accordingly, the present inventors have studied certain components that do not inhibit amplification of the target nucleic acid and optimal concentrations thereof and developed a cell lysis composition that exhibits improved performance and analytical sensitivity for target nucleic acid amplification. The cell lysis composition developed by the present inventors allows cell lysates to be directly used in a target nucleic acid amplification reaction without purification of the target nucleic acids, thereby reducing the time and cost required for the target nucleic acid amplification reaction.

[0014] 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.

[0015]

[0016] The present inventors endeavored not only to reduce the time and cost required for the conventional nucleic acid amplification reaction, but also to improve performance and analytical sensitivity. As a result, the present inventors have studied certain components that do not inhibit amplification of the target nucleic acid and optimal concentrations thereof and developed a cell lysis composition that exhibits improved performance and analytical sensitivity for target nucleic acid amplification. The cell lysis composition developed by the present inventors allows cell lysates to be directly used in a target nucleic acid amplification reaction without purification of the target nucleic acids, thereby reducing the time and cost required for the target nucleic acid amplification reaction.

[0017] Accordingly, it is an object of the present disclosure to provide a composition for lysing cells in a biological sample.

[0018] It is another object of the present disclosure to provide a method for detecting a target nucleic acid from a biological sample using the composition.

[0019] Other objects and advantages of the present disclosure will become more apparent from the following detailed description together with the appended claims and drawings.

[0020]

[0021] In an aspect of the present disclosure, there is provided a composition for lysing cells in a biological sample, the composition comprising: (a) guanidine hydrochloride (GuHCl); (b) cetyltrimethylammonium bromide (CTAB); and (c) polyethylene glycol (PEG).

[0022] In certain embodiments, the GuHCl is included in an amount of about 30 to about 300 mM.

[0023] In certain embodiments, the CTAB is included in an amount of about 0.05 to less than about 0.5 wt%, based on the total weight of the composition.

[0024] In certain embodiments, the PEG is included in an amount of about 5 to about 80 wt%, based on the total weight of the composition.

[0025] In certain embodiments, the PEG has a molecular weight of 200 to 1000 Da.

[0026] In certain embodiments, the composition further comprises a cationic, anionic, or nonionic detergent.

[0027] In certain embodiments, the anionic detergent is n-lauroylsarcosine (NLS).

[0028] In certain embodiments, the NLS is included in an amount of about 0.05 to less than about 1 wt%, based on the total weight of the composition.

[0029] In certain embodiments, the nonionic detergent is polyoxyethylene sorbitan monolaurate (Tween 20).

[0030] In certain embodiments, the Tween 20 is included in an amount of about 5 to about 15 wt%, based on the total weight of the composition.

[0031] In certain embodiments, the composition further comprises a chelating agent or a buffer.

[0032] In certain embodiments, the chelating agent includes ethylenediaminetetraacetic acid (EDTA).

[0033] In certain embodiments, the EDTA is included in an amount of about 0.1 to about 10 mM.

[0034] In certain embodiments, the buffer is tris(hydroxymethyl)aminomethane (Tris).

[0035] In certain embodiments, the Tris is included in an amount of about 1 to about 50 mM.

[0036] In certain embodiments, the composition has a pH of about 8 or greater.

[0037] In certain embodiments, the biological sample is a swab, saliva, or a mixture thereof obtained from the subject.

[0038] In certain embodiments, the swab is a throat swab or nasal swab.

[0039] In certain embodiments, the cells in the biological sample are derived from a bacterium 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,Arcanobacterium haemolyticum, and combinations thereof.

[0040] In another aspect of the present disclosure, there is provided a method for detecting a target nucleic acid from a biological sample, the method comprising: (a) mixing a biological sample with the composition as described above to lyse cells in the biological sample; (b) mixing an aliquot of the lysate with a nucleic acid amplification reagent to perform an amplification reaction; and (c) detecting a product amplified in the amplification reaction.

[0041] In certain embodiments, the method does not include additional extraction of the target nucleic acid.

[0042] In certain embodiments, the mixing in step (a) comprises incubating at 25℃ to 100℃ for 5 to 15 minutes.

[0043] In certain embodiments, the amplification reaction is PCR, real-time PCR, or Loop Mediated Isothermal Amplification (LAMP).

[0044]

[0045] The features and advantages of the present invention are summarized as follows:

[0046] (a) Since the method according to the present disclosure allows nucleic acids to be obtained without purification thereof, it is possible to shorten the time required for various nucleic acid-based diagnosis, research, and the like.

[0047] (b) The method according to the present disclosure simplifies the nucleic acid extraction into one-step process using one reagent, thereby avoiding the need for equipment, reagents, and tools used in conventional complex extraction process.

[0048] (c) The composition according to the present disclosure does not inhibit a nucleic acid amplification reaction, and thus can be used to detect a target nucleic acid with high performance and analytical sensitivity.

[0049] (d) The composition according to the present disclosure simplifies the nucleic acid extraction process to ameliorate experimenter fatigue, and can be used in large hospitals, testing institutions, and research institutes that perform molecular diagnosis of large quantities of samples.

[0050]

[0051] I. Definition

[0052] Hereinafter, the terms used in connection with the present invention are defined.

[0053] As used herein, the phrase "a composition for lysing cells" or "a cell lysis composition" refers to a composition that serves to lyse and release cells of an organism that includes nucleic acids,e.g., DNA or RNA. Specifically, the composition for lysing cells, upon contact with a biological sample obtained from a subject, destroys cells of an organism such as an animal, a plant, a yeast, a bacterium, a virus, etc., which is present in the sample, thereby releasing nucleic acids contained within it to the outside.

[0054] Conventionally, in order to extract nucleic acids from cells of an organism present in a biological sample, a series of steps have been performed, including lysis of cells, binding, washing, purification, and elution of nucleic acids. Reagents have been developed for optimized performance of each step in nucleic acid extraction, including, but not limited to, lysis buffers, binding buffers, wash buffers, elution buffers, and the like.

[0055] The "composition for lysing cells" according to the present disclosure is similar to conventional lysis buffers known in the art in that it serves to lyse cells. However, since the components in the conventional lysis buffers are likely to inhibit a nucleic acid amplification reaction such as PCR, the resulting lysates cannot be directly used in the nucleic acid amplification reaction without purification, whereas the components in the cell lysis composition according to the present disclosure have no inhibitory effect against a nucleic acid amplification reaction and thus can be directly used in the reaction without purification. In this regard, the "composition for lysing cells" according to the present disclosure may also be referred to as a "direct lysis composition", a "direct lysis buffer", a "direct lysis reagent", or a "direct lysis solution". Alternatively, the "composition for lysing cells" according to the present disclosure may also be referred to as an "extraction-free composition", an "extraction-free buffer", an "extraction-free reagent", or an "extraction-free solution", because it allows nucleic acids to be obtained without nucleic acid extraction, in particular, nucleic acid purification.

[0056] 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 cell.

[0057] 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.

[0058] As used herein, the term "biological sample" refers to an analyte obtained from a subject, which contains or is suspected of containing a nucleic acid to be detected. Examples of such biological samples 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 one embodiment, the biological sample is a swab, saliva, or a mixture thereof, collected from the subject. In certain embodiments, examples of the swab include, without limitation, a throat swab or a nasal swab, such as nasopharyngeal swab. The sample may be used interchangeably with "specimen" herein.

[0059] As used herein, the term "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" refers to a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer, and may include a natural nucleotide analogue, a non-natural nucleotide, or a modified nucleotide, which may 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 to be detected. The target nucleic acid may be hybridized with a primer or probe used in a nucleic acid amplification reaction.

[0061]

[0062] II. Composition for lysing cells

[0063] Although various nucleic acid purification methods have been developed to extract nucleic acid from a biological sample, these are complex due to the need for many processing steps and various devices depending on the reagents, conditions, and methods applied. In addition, these methods are time-consuming and often unreliable, as the efficiency of nucleic acid extraction depends on the researcher's experience and expertise.

[0064] Accordingly, the present inventors have developed a composition capable of lysing cells, which has no inhibitory effect against a nucleic acid amplification reaction and can be directly used for the nucleic acid amplification reaction without nucleic acid extraction. In particular, nucleic acids obtained using the composition developed by the present inventors exhibit higher performance and analytical sensitivity in subsequent nucleic acid amplification reactions than those obtained by conventional nucleic acid extraction processes.

[0065] According to an aspect of the present disclosure, there is provided a composition for lysing cells in a biological sample, the composition comprising: (a) guanidine hydrochloride (GuHCl); (b) cetyltrimethylammonium bromide (CTAB); and (c) polyethylene glycol (PEG).

[0066] The composition for lysing cells 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.

[0067] Hereinafter, each component included in the composition of the present invention and the optimum concentration thereof will be described.

[0068]

[0069] Guanidine hydrochloride (GuHCl)

[0070] Guanidine hydrochloride (GuHCl), which is 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.

[0071] In the method according to the present disclosure, GuHCl serves to denature proteins and to lyse cells.

[0072] 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.

[0073] 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.

[0074] In certain embodiments, the GuHCl is included in the composition in an amount of about 100 mM.

[0075] The GuHCl may be included in an amount of about 0.3 to about 3 wt%, based on the total weight of the composition.

[0076] 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.

[0077] In certain embodiments, the GuHCl is included in an amount of about 1.0 wt%, based on the total weight of the composition.

[0078] 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.

[0079]

[0080] Cetyltrimethylammonium bromide (CTAB)

[0081] 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.

[0082] 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.

[0083] 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 Examples according to the present disclosure, 0.5 wt% or more of CTAB exhibits an inhibitory activity against a nucleic acid amplification reaction.

[0084] 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.

[0085] In certain embodiments, the CTAB is included in an amount of about 0.1 wt%, based on the total weight of the composition.

[0086] The lysates obtained using a composition comprising CTAB exhibit low Ct values,i.e., high performance in a nucleic acid amplification reaction.

[0087]

[0088] Polyethylene glycol (PEG)

[0089] 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.

[0090] 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 according to 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.

[0091] 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.

[0092] In certain embodiments, the PEG is included in an amount of 60 wt%, based on the total weight of the composition.

[0093] 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).

[0094] In certain embodiments, the PEG has a molecular weight of 200 to 1,000 Da.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102]

[0103] As described above, the composition for lysing cells according to the present disclosure comprises a combination of GuHCl, CTAB, and PEG.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] In certain embodiments, the cell lysis composition according to the present disclosure comprises:

[0108] (a) about 100 mM of GuHCl;

[0109] (b) about 0.1 wt% of CTAB; and

[0110] (c) about 60 wt% of PEG200.

[0111]

[0112] Detergent

[0113] The cell lysis composition according to the present disclosure may further comprises a cationic, anionic, or nonionic detergent.

[0114] In an embodiment, the cationic detergent is selected from the group consisting of benzalkonium chloride (BAC), benzethonium chloride (BEC), methylbenzethonium, and cetylpyridinium chloride.

[0115] In one embodiment, the anionic detergent is n-lauroylsarcosine (NLS).

[0116] 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.

[0117] 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 according to the present disclosure, 1 wt% or more of NLS exhibits inhibitory activity against a nucleic acid amplification reaction.

[0118] 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.

[0119] In certain embodiments, the NLS is included in an amount of 0.05% by weight, based on the total weight of the composition.

[0120] In an embodiment, the non-ionic detergent is polyoxyethylene sorbitan monolaurate (Tween 20).

[0121] 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.

[0122] 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 according to the present disclosure, 20 wt% or more of Tween 20 exhibits inhibitory activity against a nucleic acid amplification reaction.

[0123] 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.

[0124] In certain embodiments, the Tween 20 is included in an amount of 5 wt%, based on the total weight of the composition.

[0125]

[0126] Chelating agent

[0127] The cell lysis composition according to the present disclosure may further include a chelating agent.

[0128] In an embodiment, the chelating agent includes ethylenediaminetetraacetic acid (EDTA).

[0129] 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 DNAase 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.

[0130] The EDTA may be included in the composition in an amount of about 0.1 to about 10 mM.

[0131] 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.

[0132] In certain embodiments, the EDTA is included in the composition in an amount of about 1 mM.

[0133] The EDTA may be included in an amount of about 0.02 to about 2 wt%, based on the total weight of the composition.

[0134] 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.

[0135]

[0136] Buffer

[0137] The cell lysis composition according to the present disclosure may further comprise a buffer.

[0138] In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris).

[0139] 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.

[0140] The Tris may be included in the composition in an amount of about 1 to about 50 mM.

[0141] 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.

[0142] In certain embodiments, the Tris is included in the composition in an amount of about 10 mM.

[0143] 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.

[0144] 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.

[0145] In certain embodiments, the Tris is included in an amount of about 0.1 wt%, based on the total weight of the composition.

[0146]

[0147] 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.

[0148]

[0149] In certain embodiments, the cell lysis composition according to the present disclosure comprises:

[0150] (a) about 100 mM of GuHCl;

[0151] (b) about 0.1 wt% of CTAB;

[0152] (c) about 60 wt% of PEG200;

[0153] (d) about 0.05 wt% of NLS;

[0154] (e) about 5 wt% of Tween 20;

[0155] (f) about 1 mM of EDTA; and

[0156] (g) about 10 mM of Tris,

[0157] wherein the composition has a pH of about 8.

[0158] 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.

[0159]

[0160] The cell lysis composition according to the present disclosure is used to lyse cells in a biological sample.

[0161] The cells in the biological sample may be derived from various organisms, particularly pathogens, such as bacteria or viruses.

[0162] In an embodiment, the cells in the biological sample are derived from a bacterium 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,Arcanobacterium haemolyticum, and combinations thereof.

[0163] The cell lysis composition according to the present disclosure is used to separate nucleic acids from a biological sample taken from a subject who is suffering from or at high risk of developing pharyngitis, in order to determine the presence of causative agents.

[0164] 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, various complications such as septic pharyngitis and streptococcal toxic shock syndrome may occur. It is required to accurately detect the causative agents in order to minimize complications caused by pharyngitis and to reduce the use of unnecessary antibiotics.

[0165] The major causative agents of the pharyngitis are β-hemolyticStreptococcus,Arcanobacterium haemolyticum,etc., and examples of the β-hemolyticStreptococcusincludeStreptococcus pyogenesbelonging to Group AStreptococcus(GAS), andStreptococcus dysgalactiae subsp. equisimilisandStreptococcus dysgalactiae subsp. dysgalactiaebelonging to Group CStreptococcus(GCS) or Group GStreptococcus(GGS).

[0166] The GAS is gram-positive bacteria of the genusStreptococcusthat exhibits β-hemolysis and has the A, C, and G antigens according to the Lancefield classification.

[0167] The GCS or GGS is gram-positive bacteria of the genusStreptococcusthat exhibit β-hemolysis and has C and G antigens according to the Lancefield classification.

[0168] Subjects diagnosed as infected with GAS, GCS, or GGS are treated with antibiotics such as penicillin, amoxillin, or benzathine, and if having an allergy to penicillin, are treated with antibiotics such as cephalexin, cepadroxyl, clindamycin, azithromycin, or clarithromycin.

[0169] Meanwhile,Arcanobacterium hamolyticumis a species of bacteria classified as Gram-positiveBacillus. Subjects diagnosed as infected withArcanobacterium haemolyticumare treated with an antibiotic such as erythromycin, azithromycin, gentamicin, or clindamycin.

[0170] 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, their distinguishment helps prevent abuse of antibiotics such as penicillin and prescribe correct treatments.

[0171] Isolation of nucleic acids using the cell lysis composition according to the present disclosure and subsequent amplification of three target nucleic acids enable the presence of the three pathogens to be accurately distinguished.

[0172] 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 of a patient with pharyngitis. Conventional nucleic acid amplification reactions require a complex process of purifying and extracting nucleic acids from a biological sample, whereas the cell lysis composition according to the present disclosure enables a nucleic acid to be separated from a biological sample only by a simple incubation, and thus a lysate obtained using the cell lysis composition according to the present disclosure can be directly applied to a nucleic acid amplification reaction.

[0173]

[0174] III. Method for detecting target nucleic acid

[0175] According to another aspect, there is provided a method for detecting a target nucleic acid from a biological sample, the method comprising: (a) mixing a biological sample with the composition as described above to lyse cells in the biological sample; (b) mixing an aliquot of the lysate with a nucleic acid amplification reagent to perform an amplification reaction; and (c) detecting a product amplified in the amplification reaction.

[0176]

[0177] Step (a): Lysing cells using the composition according to the present disclosure

[0178] In step (a), the biological sample is mixed with the composition according to an embodiment of the present invention. The mixing allows for lysis of the cells in the biological sample and release of the nucleic acids present in the cells out of the cells.

[0179] The mixing may be performed, for example, by (i) immersing a sample taken from a subject in a transport medium, and then mixing a portion of the transport medium with the cell lysis composition as described above, or (ii) directly mixing the sample with the cell lysis composition.

[0180] In the case of mixing a sample in a transport medium with the cell lysis composition, the sample taken may be immersed in a transport medium, and a portion of the transport medium containing the sample is mixed with the cell lysis composition. The transport medium refers to a medium for preservation that allows maintaining the integrity of a sample, for example, the cell integrity of virus or bacteria.

[0181] 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 from SG medical, Inc.

[0182] In addition, 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.

[0183] In addition, the transport medium may not include any cell lysis component. In addition, the transport medium may be a liquid medium.

[0184] According to an embodiment, in step (a), the mixing ratio of the cell lysis composition to the transport medium containing the sample may be 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 (v / v). Such mixing ratio may improve the performance of the nucleic acid isolation using the cell lysis composition and then the amplification of the isolated nucleic acid.

[0185] According to an embodiment, after preparing the mixture of the transport medium containing the sample and the cell lysis composition in step (a), the mixture may be incubated at 25℃ to 100℃ for 5 minutes to 15 minutes, 5 minutes to 12 minutes, 5 minutes to 10 minutes, 5 minutes to 7 minutes, 7 minutes to 15 minutes, 7 minutes to 12 minutes, 7 minutes to 10 minutes, 10 minutes to 15 minutes, or 10 minutes to 12 minutes. During the incubation time, the sample is sufficiently contacted with the cell lysis composition so that the nucleic acid can be released out of the cell.

[0186] In the case of directly mixing a sample with the cell lysis composition, the sample taken may be immersed and stored in the cell lysis composition for 10 minutes to 14 days.

[0187] As used herein, the term "vessel" refers to a space for accommodating materials such as a sample or reagent. The material generally comprises a solution. Herein, a space which accommodates materials used for sample preparation and sample analysis may be referred to as "container" or "carrier". These terms can be used interchangeably, but may be selectively used depending on the device, form, or internal accommodation material.

[0188] The storage may allow for maintaining the integrity of the sample for,e.g., 20 minutes or more, 1 hour or more, 2 hours or more, 5 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 7 days or more, 10 days or more, 11 days or more, or 14 days. Since the cell lysis composition according to the present invention can serve not only as a transport medium but also to extract nucleic acids, a sample mixed with the cell lysis composition according to an embodiment of the present invention can be immediately subjected to a nucleic acid amplification reaction without an additional processing such as nucleic acid extraction, purification, and the like.

[0189] Thus, the method of the present disclosure does not include additional extraction of the target nucleic acid. Specifically, the method of the present disclosure does not include purifying the target nucleic acid, such as binding the target nucleic acid to a solid support, washing of the target nucleic acid, or eluting of the target nucleic acid, after cell lysis.

[0190] The step (a) of the present method yields a lysate of cells, and the lysate is immediately subjected to the nucleic acid amplification reaction of step (b).

[0191]

[0192] Step (b): Performing an amplification reaction

[0193] In step (b), an aliquot of the lysate is mixed with a nucleic acid amplification reagent to perform an amplification reaction.

[0194] As used herein, "a nucleic acid amplification reagent" refers collectively to a mixture of components used to amplify nucleic acids. The components of the nucleic acid amplification reagent may vary depending on the type of the amplification reaction.

[0195] Herein, the nucleic acid amplification reaction may be PCR, real-time PCR, or loop mediated isothermal amplification (LAMP). In addition to the above-mentioned amplification reaction, various amplification reactions may be performed.

[0196] 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 (TMA; 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., Biotechnology 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).

[0197] In an embodiment, the amplification of the target nucleic acid may be performed by a nucleic acid amplification method, for example, a PCR method, which accompanies a change in temperature. In the nucleic acid amplification method, cycles including denaturing step, primer annealing step, and extension (or amplification) step may be repeated several times or several tens of times.

[0198] In an embodiment, the amplification of the target nucleic acid may be performed by a nucleic acid amplification method that does not accompany a change in temperature,i.e., an isothermal amplification method. Examples of isothermal amplification reactions 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).

[0199] Although PCR is described as an example of a target nucleic acid amplification method throughout the specification, those skilled in the art will recognize that various nucleic acid amplification reactions other than PCR may be applied.

[0200] According to an embodiment of the present disclosure, when the target nucleic acid is RNA, the amplification reaction may include a reverse transcription reaction step. Details thereof are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Noonan, K.F. et al., Nucleic Acids Res. 16:10366 (1988).

[0201] A reverse transcription reaction is a reaction in which cDNA is synthesized from RNA using a reverse transcriptase.

[0202] As used herein, the term "reverse transcriptase", which is also referred to as RNA-dependent DNA polymerase, refers to an enzyme that catalyzes the synthesis of DNA complementary to RNA as a template.

[0203] In the present disclosure, the reverse transcriptase may be a reverse transcriptase derived from various sources, including, without limitation, Avian Myeloblastosis Virus-derived Reverse Transcriptase (AMV RTase), Murine Leukemia Virus-derived Reverse Transcriptase (MuLV RTase), and Rous-Associated Virus 2 Reverse Transcriptase (RAV-2 RTase).

[0204] Reverse transcriptase requires primers to synthesize cDNA from RNA template. The primers used in the reverse transcription reaction include three types of primers: (i) an oligo dT primer which is annealed to poly A tail of mRNA and synthesizes cDNA from the 3'-end, (ii) a random primer which is made of a random nucleotide having a size of 6 to 9 nt and starts synthesis in all regions of RNA, and (iii) a target specific primer which synthesizes only target cDNA.

[0205] According to an embodiment of the present disclosure, the nucleic acid amplification reagent may include a reagent for reverse transcription reaction.

[0206] According to an embodiment of the present disclosure, the reagent for reverse transcription reaction may include a reverse transcription polymerase and / or a reverse transcription primer.

[0207] According to an embodiment of the present disclosure, the reverse transcription primer may be an oligo dT-primer, a random primer, or a target specific primer.

[0208] According to an embodiment of the present disclosure, the reverse transcription primer may be a target specific primer.

[0209] According to an embodiment of the present disclosure, the target-specific primer may be used to synthesize cDNA in a reverse transcription reaction, and then used as a primer pair or a primer set in combination with other primers for amplification of the synthesized cDNA in a target nucleic acid amplification reaction.

[0210] In the present disclosure, the reagent for the reverse transcription reaction may optionally include components required for performing the reverse transcription reaction, such as a buffer and deoxyribonucleotide triphosphates (dNTPs). The optimum amount of components used in a specific reaction can be readily determined by those skilled in the art who are aware of the advantages of the present disclosure. The components of the reagent for reverse transcription reaction may be present in separate containers or in a single container.

[0211] If cells in the biological sample contain DNA rather than RNA, the reverse transcription reaction is not required.

[0212] The nucleic acid amplification reagent used in the method of the present invention includes a set of oligonucleotides for detecting a target nucleic acid.

[0213] The set of oligonucleotides for detecting the target nucleic acid refers to a set of oligonucleotides specific to the target nucleic acid.

[0214] When there are a plurality of target nucleic acids, the nucleic acid amplification reagent may include a plurality of sets of oligonucleotides. A set of oligonucleotides is specific to a target nucleic acid. The phrase "a set of oligonucleotides is specific to a target nucleic acid" means that the set of oligonucleotides contributes to detecting one target nucleic acid but not to detecting other target nucleic acids. In addition, the phrase "a set of oligonucleotides is specific to a target nucleic acid" means that the set of oligonucleotides interacts with one target nucleic acid but does not interact with other target nucleic acids. The phrase is not interpreted to mean that all oligonucleotides in the set necessarily hybridizes to the target nucleic acid.

[0215] The set of oligonucleotides used in the present invention comprises: (i) an amplification 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 amplification oligonucleotide and the signaling oligonucleotide are the same or different.

[0216] As an example, the set of oligonucleotides may include an oligonucleotide that specifically hybridizes to a target nucleic acid as an amplification oligonucleotide or a signaling oligonucleotide (e.g., a probe and a primer); may include a capture oligonucleotide that specifically hybridizes to a fragment when the probe or the primer hybridized to the target nucleic acid is cleaved to release the fragment; may include an oligonucleotide that specifically hybridizes to an extension strand when the fragment hybridized to the capture oligonucleotide is extended to form the extension strand; may include an oligonucleotide that specifically hybridizes to the capture oligonucleotide; or may include a combination of the foregoing.

[0217] As used herein, "an amplification oligonucleotide" refers to an oligonucleotide that serves to amplify a target nucleic acid of interest.

[0218] In one embodiment, the amplifying oligonucleotide is a "primer" known in the art. As used herein, the term "primer" refers to an oligonucleotide that can act as an initiation point of synthesis under conditions in which synthesis of a primer extension product complementary to a target nucleic acid (template) is induced,i.e., under conditions of the presence of a nucleotide and a polymerization agent such as a DNA polymerase, and suitable temperature and pH. The primer should be long enough to prime the synthesis of the extension product in the presence of a polymerization agent. The suitable length of a primer is determined according to a number of factors, such as temperature, application, and source of the primer.

[0219] 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 amplification oligonucleotide may be an oligonucleotide having a structure known in the art, and may be synthesized by a method known in the art.

[0220] As used herein, the term "signaling oligonucleotide" refers to an oligonucleotide involved in the generation of detectable signals. According to an embodiment of the present invention, the signaling oligonucleotide includes an oligonucleotide involved in actual signal generation. For example, hybridization or non-hybridization of a signaling oligonucleotide with another oligonucleotide (e.g., a target nucleic acid or an oligonucleotide comprising a nucleotide sequence complementary to the signaling oligonucleotide) determines the generation of detectable signals.

[0221] 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 including a region or regions substantially complementary to a target nucleic acid. According to one embodiment of the invention, the 3-end of the probe is "blocked" to prevent its extension. The blocking can be accomplished according to conventional methods. For example, the blocking may be carried out by adding a chemical moiety such as a biotin, a label, a phosphate group, an alkyl group, a non-nucleotide linker, a phosphorothioate, or an alkane-diol residue to the 3'-hydroxyl group of the last nucleotide. Alternatively, the blocking may be carried out by removing the 3'-hydroxyl group of the last nucleotide or by using a nucleotide without a 3'-hydroxyl group, such as a dideoxynucleotide.

[0222] The signaling oligonucleotide herein has at least one fluorescent label linked thereto, and the fluorescent label on the signaling oligonucleotide generates a fluorescent signal.

[0223] According to an embodiment of the present invention, the signaling oligonucleotide may be composed of at least one oligonucleotide. According to an embodiment of the present invention, when the signaling oligonucleotide is composed of a plurality of oligonucleotides, the signaling oligonucleotide may have a label in various ways. For example, one of the plurality of oligonucleotides may have at least one label, or all the plurality of oligonucleotides may have at least one label.

[0224] According to the present invention, the amplification oligonucleotide and the signaling oligonucleotide in the set of oligonucleotides are the same or different.

[0225] The expression "the amplification oligonucleotide and the signaling oligonucleotide are the same" means that an oligonucleotide serves as an amplification oligonucleotide which amplifies the target nucleic acid as well as a signaling oligonucleotide which generates a signal in the presence of a target nucleic acid. As an example, an oligonucleotide having a fluorescent label may be hybridized with a target nucleic acid to be extended and generate a signal.

[0226] The expression "the amplification oligonucleotide and the signaling oligonucleotide are different" means that two different oligonucleotides are used.

[0227] According to the present disclosure, a set of oligonucleotides comprising a signaling oligonucleotide may generate a signal by one of methods commonly known in the art.

[0228] In one embodiment, the set of oligonucleotides generates a signal by formation of a duplex from a signaling oligonucleotide or dissociation of the formed duplex.

[0229] Particularly, the signal is generated by a duplex formed between a target nucleic acid and a signaling oligonucleotide that is specifically hybridized with the target nucleic acid.

[0230] Generation of signals by a duplex formed between a target nucleic acids and a signaling oligonucleotide may be accomplished by a variety of 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 CB, et al., Journal of the American Chemical Society, 126:4550-4556 (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).

[0231] According to an embodiment, the set of oligonucleotides generates a signal by cleavage of a mediation oligonucleotide hybridized to a target nucleic acid which occurs depending on the presence of the target nucleic acid, followed by formation of a duplex between a cleavage product and a signaling oligonucleotide or dissociation of the formed duplex.

[0232] A signal generated by a duplex formed in a manner dependent on cleavage of the mediation oligonucleotide may refer to a signal by PTO cleavage and extension (PTOCE) method (WO 2012 / 096523), PTO cleavage and extension-dependent signaling oligonucleotide hybridization (PCE-SH) method (WO 2013 / 115442), PTO cleavage and extension-dependent non-hybridization (PCE-NH) method (WO 2014 / 104818), or methods disclosed in U.S. Pat. Nos. 11,034,997, 11,028,433, and 10,590,469, and U.S. Patent Publication No. 2020-0048682.

[0233] According to an embodiment, the set of oligonucleotides generates a signal by hybridization of a signaling oligonucleotide with a target nucleic acid that occurs depending on the presence of the target nucleic acid, followed by cleavage of a duplex.

[0234] The generation of signals by hybridization of the signaling oligonucleotide with a target nucleic acid, followed by cleavage may be accomplished by various methods including TaqMan probe methods (U.S. Pat. Nos. 5,210,015 and 5,538,848).

[0235] In particular, the generation of signals is accomplished by cleavage of a signaling oligonucleotide in a manner that is dependent upon cleavage of a mediation oligonucleotide that is specifically hybridized to the target nucleic acid.

[0236] The generation of signals by cleavage of the signaling oligonucleotide in a manner dependent on cleavage of the mediation oligonucleotide may be accomplished by various methods including Invader analysis (U.S. Pat. No. 5,691,142), PTO cleavage and extension-dependent cleavage (PCEC) method (WO 2012 / 134195), and a method described in U.S. Pat. No. 7,309,573.

[0237] Fluorescent labels useful in the present invention include various fluorescent labels known in the art. For example, fluorescent labels useful in the present invention include single fluorescent labels, and interactive dual fluorescent labels.

[0238] The single fluorescent label provides different signals (e.g., different signal intensities) depending on whether the single fluorescent label is on a double strand or a single strand. Preferred types and binding sites of a single fluorescent label used in the present invention are disclosed in 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 the oligonucleotide by various methods. For example, the label is linked to the probe via a spacer containing carbon atoms (e.g., 3-carbon spacer, 6-carbon spacer, or 12-carbon spacer).

[0239] As a representative example of the interactive fluorescent labeling system, a fluorescence resonance energy transfer (FRET) fluorescent labeling system includes a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor may be fluorescent, but the energy acceptor may be fluorescent or non-fluorescent. In another form of an interactive label system, the energy donor is non-fluorescent,e.g., a chromophore and the energy acceptor is fluorescent. In another form of an interactive label system, the energy donor is luminescent,e.g., bioluminescent, chemiluminescent, or electrochemiluminescent and the acceptor is fluorescent. The interactive label system includes dual labels based on "contact-mediated quenching" (Salvatore et al., Nucleic Acids Research, 2002 (30) no. 21 e122 and Johansson et al., J. AM. CHEM. SOC 2002 (124) pp 6950-6956). The interactive labeling system includes any labeling system as long as it induces a signal change by interaction between at least two molecules (e.g., dyes).

[0240] Reporter molecules and quencher molecules useful in the present invention may include any molecule known in the art. The examples are as follows: Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568), BODIPY564 / 570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), Rphycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). The number of parentheses is a maximum emission wavelength in nanometers.

[0241] Preferably, the reporter molecule and quencher molecule include JOE, FAM, TAMRA, ROX and fluorescein-based label.

[0242] Suitable fluorescent molecules and suitable reporter-quencher pairs are disclosed in Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene Oreg., 1996); U.S. Pat. Nos. 3,996,345 and 4,351,760.

[0243] In the present invention, a non-fluorescent quencher molecule (e.g., a black quencher or a dark quencher) capable of quenching fluorescence of a wide wavelength or a specific wavelength may be used.

[0244] In a signaling system comprising a reporter molecule and a quencher molecule, the reporter molecule comprises a donor of FRET and the quencher molecule comprises an acceptor of FRET. For example, fluorescein dye is used as a reporter molecule and rhodamine dye is used as a quencher molecule.

[0245] The amplification reaction in step (b) as described above allows for amplification of the target nucleic acid,i.e., generation of amplified products, leading to generation of detectable signals.

[0246]

[0247] (c): Detection of an amplified product

[0248] Subsequently, a product amplified in the amplification reaction of step (b) is detected in step (c).

[0249] The detection may be performed by one of methods commonly known in the art.

[0250] In one embodiment, the detection is carried out by analysis of an amplification curve obtained during the amplification reaction of step (b).

[0251] In another embodiment, the detection is carried out by analysis of a melting curve obtained by the amplification reaction of step (b), followed by the melting of an amplified product.

[0252] In another embodiment, the detection is carried out by analysis of an amplification curve obtained by the melting of an amplified product, followed by the hybridization of the melted product.

[0253] As used herein, the term "melting analysis" refers to a method of determining the presence of a target nucleic acid by signals which is obtained by amplifying the target nucleic acid and then melting the amplified product. The melting analysis includes melting curve analysis, melting pattern analysis, and melting peak analysis. Preferably, the melting analysis is melting curve analysis.

[0254] Melting curve or amplification curve can be obtained by conventional techniques as disclosed in,e.g., U.S. Pat. Nos. 6,174,670 and 5,789,167, Drobyshev et al., Gene 188: 45 (1997); Kochinsky and Mirzabekov Human Mutation 19:343 (2002); Livehits et al., J. Biomol. Structure Dynam. 11:783 (1994); and Howell et al., Nature Biotechnology 17:87 (1999).

[0255] The detection is carried out by measuring signals generated in the presence of amplified products.

[0256] A signal refers to one generated during an amplification reaction, and the generation of a signal includes "generation or extinguishment" of a signal and "increase or decrease" in a signal from a label. In the present disclosure, the generation of a signal means the generation of a significant signal,i.e., a signal indicative of the presence of a target nucleic acid. For example, a significant signal,i.e., a signal indicative of the presence of a target nucleic acid, refers to a signal having an intensity that exceeds the intensity of a background signal or a signal in the absence of the target nucleic acid, or to a signal having an intensity obtained by subtracting the intensity of a background signal or a signal in the absence of the target nucleic acid from the intensity of a generated signal.

[0257] The nucleic acid amplification reaction herein yields an amplification curve that indicates amplification of a target nucleic acid in the presence of the target nucleic acid in a sample.

[0258] Herein, an amplification curve refers to a curve obtained by plotting a data set obtained by an amplification reaction for a target nucleic acid. In one embodiment, the data set includes a plurality of data points, each including a cycle number and a signal value.

[0259] As used herein, the term "signal value" refers to a value obtained by quantifying the level of a signal (e.g., signal intensity) measured in a cycle of a nucleic acid amplification reaction according to a certain scale, or a modified value thereof. The modified value may include a mathematically processed signal value of the measured signal value. An example of a mathematically processed signal value of an actually measured signal value (i.e., a signal value of a raw data set) may include a logarithmic value or derivatives.

[0260] The term "data point" as used herein refers to a coordinate value including a cycle and a signal value. The term "data" means all information constituting a data set. For example, each of the cycle and the signal value in an amplification curve is data.

[0261] The data points obtained by the amplification reaction may be represented by coordinate values in a two-dimensional orthogonal coordinate system. In the coordinate value, the X-axis represents the number of a cycle, and the Y-axis represents a signal value measured or processed in a corresponding cycle.

[0262] As used herein, the term "data set" refers to a collection of the data points. For example, the data set may be a set of data points directly obtained through an amplification reaction performed in the presence of a set of oligonucleotides specific to a target nucleic acid, or may be a modified data set thereof. The data set may be all or any portion of a plurality of data points obtained by an amplification reaction or modified data points thereof.

[0263]

[0264] In the present disclosure, the detection of the amplified products may be carried out by identifying a melting peak in a melting analysis. Alternatively, the detection of the amplified products may be carried out by identifying a significant signal in real-time PCR, for example, determining a Ct value by using a predetermined threshold, determining as a quantification cycle (Cq) value, a first derivative maximum (FDM), a second derivative maximum (SDM), or the like, as disclosed in U.S. Pat. Nos. 6,503,720, 6,783,934, 7,565,250, 8,285,489, or 10,176,293.

[0265] As an example, the existence of a melting peak corresponding to the Tm value of an amplified products in the melting curve allows for determination of the presence of the target nucleic acid, whereas no existence of the melting peak allows for determination of absence of the target nucleic acid.

[0266] As another example, when a predetermined threshold is applied to an amplification curve, the intersection between the threshold and the amplification curve allows for determination of the presence of the target nucleic acid, whereas no intersection between the threshold and the amplification curve allows for determination of the absence of the target nucleic acid.

[0267] As another example, when FDM as a Cq value is determined from an amplification curve, FDM not more than a predetermined cycle (e.g., 45 cycle, 50 cycle, etc.) allows for determination of the presence of the target nucleic acid, whereas FDM more than the predetermined cycle allows for determination of the absence of the target nucleic acid.

[0268] The method according to the present disclosure uses a cell lysis composition comprising a combination of components at optimal concentrations that exhibit excellent nucleic acid lysis performance without inhibiting a nucleic acid amplification reaction. The method according to the present disclosure can allow for detection of a target nucleic acid with improved analytical sensitivity and reduce the time required for detection of a target nucleic acid, enabling high throughput analysis.

[0269]

[0270] 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.

[0271]

[0272] EXAMPLES

[0273] Example 1: Determination of Optimal Concentration of Cell Lysis Components

[0274] 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)).

[0275] 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.

[0276] 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%.

[0277] 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.

[0278] Thereafter, each of the experimental groups and the control were incubated at 25℃ for 10 minutes to lyse the cells ofS. pyogenes.

[0279] 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.

[0280] NameSequenceSEQ ID NOspy-F5'-GCACTCGCTACTATTTCTTACCTCAA-3'1spy-R5'-GTCACAATGTCTTGGAAACCAGTAAT-3'2spy-P5'-FAM-CCGCAACTCATCAAGGATTTCTGTTACCA-BHQ1-3'3

[0281] 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.

[0282]

[0283] <1-1> GuHCl

[0284] 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.

[0285] 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

[0286] 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.

[0287] The results demonstrate that it is preferred to add GuHCl in an amount of about 30 to about 300 mM.

[0288]

[0289] <1-2> CTAB

[0290] 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.

[0291] 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

[0292] 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.

[0293] The results demonstrate that it is preferred to add CTAB in an amount of about 0.05 wt% to less than 0.5 wt%.

[0294]

[0295] <1-3> PEG200

[0296] 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.

[0297] ReactionControlPEG2005 wt%10 wt%15 wt%20 wt%25 wt%Ct Value#133.3532.9232.2932.6031.8432.59#232.2732.5632.1432.1232.2532.37#332.5432.8632.3032.2532.3232.49Average32.7232.7832.2432.3232.1432.48Experimental group - Control-0.06-0.48-0.40-0.58-0.24Standard Deviation0.560.190.090.250.260.11RFU#11180.201307.151423.391264.361318.411241.85#21192.161413.181385.731419.791270.661393.67#31199.841309.071375.361320.241266.191359.10Average1190.731343.131394.821334.801285.091331.54Experimental group - Control-152.40204.09144.0694.36140.81

[0298] ReactionControlPEG20030 wt%40 wt%50 wt%60 wt%70 wt%80 wt%Ct 값#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

[0299] 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.

[0300] The results demonstrate that 5 wt% to 80 wt% of PEG200 has no significant effect on the PCR reaction.

[0301]

[0302] <1-4> NLS

[0303] 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.

[0304] 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

[0305] 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.

[0306] The results demonstrate that it is preferred to add NLS in an amount of about 0.05 wt% to less than 1 wt%.

[0307]

[0308] <1-5> Tween 20

[0309] 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.

[0310] 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

[0311] As shown in Table 7, when Tween 20 was added at 20 wt% and 25 wt%, the RFU values were slightly decreased.

[0312] The results demonstrate that it is preferred to add Tween 20 in an amount of about 5 wt% to 15 wt%.

[0313]

[0314] Example 2: Effect of pH of Cell Lysis Composition

[0315] 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.

[0316] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] The results are shown in Table 8 below. Each result is an average of three replicates.

[0321] 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

[0322] 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.

[0323]

[0324] Example 3: Effect of CTAB and PEG combination

[0325] 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.

[0326] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] The results are shown in Table 9 below. Each result is an average of three replicates.

[0331] 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

[0332] As shown in Table 9, experimental groups 2 and 3 comprising CTAB and PEG showed lower Ct values compared to experimental group 1 comprising GuHCl and PEG, experimental group 4 comprising only PEG, and the control, indicating excellent PCR reactions of experimental groups 2 and 3 comprising CTAB and PEG.

[0333] 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.

[0334]

[0335] Example 4: Effects of GuHCl

[0336] In order to examine the effect of GuHCl on cell lysis, compositions containing GuHCl was compared with compositions not containing GuHCl.

[0337] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0338] Then, (i) a composition containing no GuHCl, a low concentration of CTAB, and PEG (Experimental Group 1), (ii) a composition containing no GuHCl, 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.

[0339] 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.

[0340] 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.

[0341] The results are shown in Table 10 below. Each result is an average of three replicates.

[0342] 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

[0343] 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.

[0344] 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.

[0345]

[0346] Example 5: Effects of PEG200

[0347] In order to examine the effect of the molecular weight of PEG on cell lysis, compositions containing PEG200 or PEG8000 were compared.

[0348] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0349] Then, (i) a composition containing PEG8000 (Experimental group 1), and (ii) a composition containing PEG200 (Experimental group 2) were prepared as experimental groups.

[0350] 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.

[0351] 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.

[0352] The results are shown in Table 11 below. Each result is an average of three replicates.

[0353] 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

[0354] 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%.

[0355] 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.

[0356]

[0357] Example 6: Effect of Tris and PEG200

[0358] In order to examine the effect of Tris and PEG200 on cell lysis, compositions containing different concentrations of Tris and PEG200 were compared.

[0359] First,Streptococcus pyogeneswas spiked in a tube of ESwab® 480C (Copan Diagnostics Inc) at a concentration of 6.02 X 106CFU / mL.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] The results are shown in Table 12 below. Each result is an average of three replicates.

[0364] 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

[0365] 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.

[0366] 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.

[0367]

[0368] Example 7: Analytical Sensitivity of Cell lysis Composition

[0369] 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.

[0370] 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.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] 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

[0375] 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.

[0376] The results are shown in Table 14 below. Each result is an average of three replicates.

[0377] 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-

[0378] 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-N / A37.03N / AN / A

[0379] 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-

[0380] 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.

[0381] 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.

[0382] 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.

[0383] In summary, 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.

[0384] 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

1.A composition for lysing cells in a biological sample, the composition comprising:(a) guanidine hydrochloride (GuHCl);(b) cetyltrimethylammonium bromide (CTAB); and(c) polyethylene glycol (PEG).2.The composition of claim 1, wherein the GuHCl is included in an amount of about 30 to about 300 mM.3.The composition of claim 1, wherein the CTAB is included in an amount of about 0.05 to less than about 0.5 wt%, based on the total weight of the composition.4.The composition of claim 1, wherein the PEG is included in an amount of about 5 to about 80 wt%, based on the total weight of the composition.5.The composition of claim 1, wherein the PEG has a molecular weight of 200 to 1000 Da.6.The composition of claim 1, further comprising a cationic, anionic, or nonionic detergent.7.The composition of claim 6, wherein the anionic detergent is n-lauroylsarcosine (NLS).8.The composition of claim 7, wherein the NLS is included in an amount of about 0.05 to less than about 1 wt%, based on the total weight of the composition.9.The composition of claim 6, wherein the nonionic detergent is polyoxyethylene sorbitan monolaurate (Tween 20).10.The composition of claim 9, wherein the Tween 20 is included in an amount of about 5 to about 15 wt%, based on the total weight of the composition.11.The composition of claim 1, further comprising a chelating agent or a buffer.12.The composition of claim 11, wherein the chelating agent includes ethylenediaminetetraacetic acid (EDTA).13.The composition of claim 12, wherein the EDTA is included in an amount of about 0.1 to about 10 mM.14.The composition of claim 11, wherein the buffer is tris(hydroxymethyl)aminomethane (Tris).15.The composition of claim 14, wherein the Tris is included in an amount of about 1 to about 50 mM.16.The composition of claim 1, wherein the composition has a pH of about 8 or greater.17.The composition of claim 1, wherein the biological sample is a swab, saliva, or a mixture thereof obtained from the subject.18.The composition of claim 17, wherein the swab is a throat swab or nasal swab.19.The composition of claim 1, wherein the cells in the biological sample are derived from a bacterium 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,Arcanobacterium haemolyticum, and combinations thereof.20.A method for detecting a target nucleic acid from a biological sample, the method comprising:(a) mixing a biological sample with the composition as described above to lyse cells in the biological sample;(b) mixing an aliquot of the lysate with a nucleic acid amplification reagent to perform an amplification reaction; and(c) detecting a product amplified in the amplification reaction.21.The method of claim 20, wherein the method does not include additional extraction of the target nucleic acid.22.The method of claim 20, wherein the mixing in step (a) comprises incubating at 25℃ to 100℃ for 5 to 15 minutes.23.The method of claim 20, wherein the amplification reaction is PCR, real-time PCR, or Loop Mediated Isothermal Amplification (LAMP).