RNA extraction method and reagent set for RNA extraction

Abstract

This RNA extraction method comprises: (1) adding an alkali metal salt and a cell wall-degrading enzyme to a sample to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, the final concentration of the alkali metal salt being 50 mM to 150 mM; (2-1) incubating the reaction solution to degrade the cell walls of microorganisms contained in the reaction solution; and (3) extracting RNA from the reaction solution after the incubation. This reagent set for RNA extraction contains: an alkali metal salt solution in which the concentration of the alkali metal salt is 50 mM or more; and a cell wall degrading enzyme.

Description

RNA extraction method and RNA extraction reagent set

[0001] This disclosure relates to an RNA extraction method and a reagent set for RNA extraction.

[0002] Cell and gene therapies generally need to be administered to patients within 48 to 96 hours of manufacture. This means that cell and gene therapies have a shelf life of only 48 to 96 hours after manufacture. Microbiological testing by culture (for example, a 14-day culture test to determine the presence of bacteria and fungi) is not applicable to pharmaceuticals with such short shelf lives. There is a need for rapid microbiological testing that is applicable to pharmaceuticals with short shelf lives.

[0003] Nucleic acid amplification testing (NAT) is a test that can rapidly detect nucleic acids and is promising as a rapid microbiological test. For example, Patent Documents 1 to 3 disclose an invention in which nucleic acids are extracted by treating microorganisms with cell wall-degrading enzymes and then performing microbiological testing using NAT.

[0004] International Publication No. 2006 / 085906, International Publication No. 2020 / 218553, International Publication No. 2019 / 030261

[0005] rRNA (ribosomal RNA) is a component of ribosomes and exists in multiple copies within cells, making it a highly sensitive target for NAT (Natural Antimicrobial Agent). Furthermore, rRNA sequences have been analyzed across a wide variety of organisms, resulting in a comprehensive sequence database. Therefore, rRNA sequences that are specific to and common to microorganisms are promising targets for microbial testing using NAT.

[0006] However, RNA is a nucleic acid that is more easily degraded than DNA (deoxyribonucleic acid). The main cause of RNA degradation is ribonuclease within cells, and when the cell wall of a microorganism is removed and RNA is extracted, the cell is rapidly destroyed and its contents are released, which may lead to the RNA being degraded by ribonuclease.

[0007] Furthermore, microorganisms differ in the resistance of their cell walls and cell membranes (e.g., resistance to degradation by cell wall-degrading enzymes, mechanical resistance to osmotic pressure) depending on the species. Therefore, in a single reaction solution, it is possible that some microorganisms may be rapidly destroyed and their RNA degraded, while other microorganisms may remain intact and their RNA cannot be extracted. To realize a single NAT microbiological test targeting RNA, it is necessary to extract RNA from all microorganisms present in the sample, but it is not easy to extract RNA from all types of microorganisms in a single reaction solution.

[0008] This disclosure is made under the circumstances described above. The object of this disclosure is to provide an RNA extraction method and a reagent set for RNA extraction that comprehensively extracts RNA from microorganisms contained in a sample, regardless of the type of microorganism.

[0009] Specific means for solving the above problems include the following embodiments: <1> A method for extracting RNA from microorganisms contained in a sample, comprising the following (1), (2-1), and (3): (1) Adding an alkali metal salt and a cell wall-degrading enzyme to a sample to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme and having a final concentration of the alkali metal salt of 50 mM to 150 mM; (2-1) Incubating the reaction solution to degrade the cell walls of microorganisms contained in the reaction solution; (3) Extracting RNA from the reaction solution after incubation. <2> The RNA extraction method according to <1>, further comprising the following (2-2) between (2-1) and (3): (2-2) Adding a proteolytic enzyme to the reaction solution after incubation to degrade the proteins contained in the reaction solution. <3> The RNA extraction method according to <1> or <2>, wherein (3) comprises degrading DNA using a deoxyribonuclease. <4> The RNA extraction method according to any one of <1> to <3>, wherein (1) comprises the following (1a) and (1b): (1a) adding an alkali metal salt to a sample and pre-incubating it; (1b) adding a cell wall-degrading enzyme to the sample after pre-incubation to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, wherein the final concentration of the alkali metal salt is 50 mM to 150 mM. <5> The RNA extraction method according to any one of <1> to <4>, wherein the alkali metal salt comprises at least one selected from the group consisting of sodium chloride, potassium chloride, and lithium chloride. <6> The RNA extraction method according to any one of <1> to <5>, wherein the cell wall-degrading enzyme comprises at least one selected from the group consisting of lysozyme, achromopeptidase, glucoamylase, and glucanase. <7> The RNA extraction method according to any one of <1> to <5>, wherein the cell wall degrading enzyme comprises lysozyme, achromopeptidase, and at least one selected from the group consisting of glucoamylase and glucanase. <8> The RNA extraction method according to any one of <2> to <7>, wherein the proteolytic enzyme comprises proteinase K.<9> The RNA extraction method according to any one of <1> to <8>, wherein the microorganism is a bacterium and / or a fungus. <10> A reagent set for extracting RNA from a microorganism contained in a sample, comprising an alkali metal salt solution having an alkali metal salt concentration of 50 mM or more, and a cell wall degrading enzyme. <11> The RNA extraction reagent set according to <10>, further comprising a proteolytic enzyme and / or deoxyribonuclease. <12> The RNA extraction reagent set according to <10> or <11>, wherein the alkali metal salt comprises at least one selected from the group consisting of sodium chloride, potassium chloride, and lithium chloride. <13> The RNA extraction reagent set according to any one of <10> to <12>, wherein the cell wall degrading enzyme comprises at least one selected from the group consisting of lysozyme, achromopeptidase, glucoamylase, and glucanase. <14> The RNA extraction reagent set according to any one of <10> to <12>, wherein the cell wall degrading enzyme comprises lysozyme, achromopeptidase, and at least one selected from the group consisting of glucoamylase and glucanase. <15> The RNA extraction reagent set according to any one of <11> to <14>, wherein the proteolytic enzyme comprises proteinase K.

[0010] According to this disclosure, an RNA extraction method and a reagent set for RNA extraction are provided, which comprehensively extract RNA from microorganisms contained in a sample, regardless of the type of microorganism.

[0011] Experiment 1: Amplification curve of RT-qPCR Experiment 2: Amplification curve of RT-qPCR Experiment 3: Correlation between alkali metal salt concentration and Ct value

[0012] The embodiments of this disclosure are described below. These descriptions and embodiments are illustrative and do not limit the scope of the embodiments.

[0013] In this disclosure, numerical ranges indicated using "~" represent a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages within this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described within this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples.

[0014] In this disclosure, "A and / or B" is synonymous with "at least one of A and B." In other words, "A and / or B" may be A alone, B alone, or a combination of A and B.

[0015] In this disclosure, the term "process" includes not only independent processes but also processes that cannot be clearly distinguished from other processes, provided that their objectives are achieved.

[0016] When referring to the amount of each component in a composition in this disclosure, if there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, it refers to the total amount of those multiple types of substances present in the composition.

[0017] Regarding substance concentration, "M" represents molar concentration, where 1M = 1 mol / L. Unless otherwise specified, "%" in relation to substance concentration refers to a mass basis.

[0018] The full spelling of abbreviations used in this disclosure is as follows: PCR: Polymerase Chain Reaction qPCR: Quantitative Polymerase Chain Reaction RT-PCR: Reverse Transcription-Polymerase Chain Reaction RT-qPCR: Reverse Transcription-Quantitative Polymerase Chain Reaction

[0019] qPCR is also called rtPCR (real-time Polymerase Chain Reaction). To avoid confusion between rt (real-time) and RT (Reverse Transcription), this disclosure will refer to it as qPCR, not rtPCR.

[0020] <Experiment> The RNA extraction method described herein was created based on the results of experiments conducted by the inventors. The experiments conducted by the inventors and their results are described below.

[0021] [Experiment 1: Examination of the effect of cell wall-degrading enzymes on fungi] Commercially available freeze-dried fungi were used: Aspergillus brasiliensis (Catalog No. 56049, bioMérieux Co., Ltd.) and Candida albicans (Catalog No. 56050, bioMérieux Co., Ltd.) (both BIOBALL standard strains for quantitative testing, 1 x 10⁻⁶). ４ CFU (Cellular Fuel Extract) was prepared. Freeze-dried bacteria were suspended in 4 mL of PBS (Phosphate Buffered Saline) to prepare bacterial suspensions for each bacterium.

[0022] A. brasiliensis fungal suspension 200 μL (5 × 10 ２ Add 0.5 mg / mL of glucoamylase to CFU / Sample, and then add 200 μL of C. albicans bacterial suspension (5 × 10⁻⁶). ２ Chitalase (a type of glucanase) at a concentration of 0.5 mg / mL was added to CFU (Cellular Fuel Extract / Sample). Each sample was incubated at 37°C for 15 minutes to decompose the fungal cell walls.

[0023] Next, the following reagents and proteinase K were added, and the mixture was incubated at 56°C for 20 minutes to decompose the proteins in the reaction solution.

[0024] • Lysis Buffer (chaotropic salt solution) 120 μL • Lytic Enhancer (surfactant mixture solution) 60 μL • Proteinase K 0.675 mg / mL

[0025] Next, nucleic acid extraction was performed using the Boom method with silica-coated magnetic beads. DNA hydrolysis with DNase I was carried out during this process. The recovered and washed magnetic beads were suspended in 100 μL of Elution Buffer (Tris-EDTA buffer), and then the magnetic beads were removed to obtain the RNA sample.

[0026] RT-qPCR was performed using RNA samples as templates, targeting conserved sequences of fungal rRNA. The amplification curves of the RT-qPCR are shown in Figure 1. The top curve in Figure 1 is the amplification curve of the control experiment with A. brasiliensis × glucoamylase (GA), and the bottom curve in Figure 1 is the amplification curve of the control experiment with C. albicans × chitalase (KA).

[0027] As is clear from Figure 1, nucleic acids were not amplified in GA(-) and KA(-), while they were amplified in GA(+) and KA(+). This indicates that the RNA samples in GA(-) and KA(-) did not contain RNA, while the RNA samples in GA(+) and KA(+) did contain RNA.

[0028] The results of Experiment 1 showed that RNA can be efficiently extracted by degrading the cell wall of fungi with cell wall-degrading enzymes.

[0029] [Experiment 2: Examination of cell wall-degrading enzyme types] Micrococcus luteus (NBRC13867, National Institute of Technology and Evaluation) was cultured and treated with sterile water in 2 x 10⁶ solutions. ５ A bacterial suspension was prepared by diluting to CFU / mL. 500 μL of bacterial suspension (1 × 10⁶) ５ One, two, or three of the following cell wall-degrading enzymes were added to CFU (Sample), and the cells were incubated at 37°C for 15 minutes to degrade the cell walls.

[0030] • Lysozyme (LS) 1 mg / mL • Achromopeptidase (AP) 200 units / mL • Glucoamylase (GA) 0.5 mg / mL

[0031] Next, the following reagents and Proteinase K were added, and the mixture was incubated at 56°C for 20 minutes to decompose the proteins contained in the reaction solution.

[0032] - Lysis Buffer 300 μL - Lytic Enhancer 150 μL - Proteinase K 0.675 mg / mL

[0033] Next, nucleic acid extraction by the Boom method using silica-coated magnetic beads was performed. During this process, DNA hydrolysis by DNase I was carried out. The recovered and washed magnetic beads were suspended in 100 μL of Elution Buffer, and then the magnetic beads were removed to obtain an RNA sample.

[0034] Using the RNA sample as a template, RT-qPCR targeting the conserved sequence of bacterial rRNA was performed. The amplification curve of RT-qPCR is shown in Figure 2, and the Ct value (Threshold Cycle) is shown in Table 1.

[0035]

[0036] As is clear from Figure 2 and Table 1, the Ct value was the lowest when three types of enzymes were used, and the more types of enzymes were used, the lower the Ct value was. It was shown that the cell wall of M. luteus was efficiently decomposed by multiple types of enzymes, and the RNA extraction efficiency was improved.

[0037] From the results of Experiment 2, it was shown that by using a combination of multiple types of cell wall degrading enzymes, the decomposition efficiency of the cell wall of microorganisms increased, and the RNA extraction efficiency was improved.

[0038] [Experiment 3: Examination of the effect of alkali metal salts] From another experiment conducted by the present inventor, it was found that E. coli is a bacterium that is easily lysed, and S. epidermidis is a bacterium that is difficult to lyse. The effect of alkali metal salts was examined using E. coli, S. epidermidis, and the fungus C. albicans.

[0039] E. coli (NBRC 3972, National Institute of Technology and Evaluation) and S. epidermidis (NBRC 12993, National Institute of Technology and Evaluation) were each cultured and diluted with sterilized water to 5 × 10 ５ CFU / mL to prepare a bacterial solution of each bacterium. C. albicans (NBRC 1594, National Institute of Technology and Evaluation) was cultured and diluted with sterilized water to 5 × 10 ４ CFU / mL to prepare a bacterial solution. 200 μL of the bacterial solution (1 × 10 ５ CFU / Sample or 1 × 10 ４ CFU / Sample) was added with the following four types of cell wall degrading enzymes and an alkali metal salt (sodium chloride, potassium chloride or lithium chloride), and incubated at 37°C for 15 minutes to decompose the cell wall.

[0040] ・ Lysozyme 1 mg / mL ・ Kitase 0.5 mg / mL ・ Glucoamylase 0.5 mg / mL ・ Achromopeptidase 200 unit / mL ・ Sodium chloride, potassium chloride or lithium chloride final concentration 0 mM to 200 mM

[0041] Next, the following reagents and Proteinase K were added, and incubated at 56°C for 20 minutes to decompose the proteins contained in the reaction solution.

[0042] ・ Lysis Buffer 120 μL ・ Lytic Enhancer 60 μL ・ Proteinase K 0.675 mg / mL

[0043] Next, nucleic acid extraction by the Boom method using silica-coated magnetic beads was performed. During the process, DNA hydrolysis by DNaseI was performed. The recovered and washed magnetic beads were suspended in 100 μL of Elution Buffer, and then the magnetic beads were removed to obtain an RNA sample.

[0044] RT-qPCR targeting the conserved sequence of bacterial rRNA or fungal rRNA was performed using the RNA sample as a template. The results are shown in Table 2, Table 3, Table 4 and Figure 3.

[0045]

[0046]

[0047]

[0048] E. coli performed best in the case where neither cell wall-degrading enzyme nor alkali metal salt was added (Ct value 21.0), and worst in the case where cell wall-degrading enzyme was added but alkali metal salt was not (Ct value 25.0). This indicates that E. coli can be lysed by the action of chaotropic salt and surfactant, and that there is little need to use cell wall-degrading enzyme. As shown in Figure 3, in the case where cell wall-degrading enzyme was added to E. coli, there was a tendency for the results to be worse (higher Ct value) as the alkali metal salt concentration decreased. It is presumed that when the alkali metal salt concentration in the reaction solution is low, the osmotic pressure difference acts significantly on the cells whose cell walls have been degraded by the enzyme, causing the cells to break down and release their contents, and the RNA to be degraded by the action of ribonuclease.

[0049] In the case of S. epidermidis, the Ct value was 33.0 in the case where neither cell wall-degrading enzyme nor alkali metal salt was added, while the Ct value was 29.0 in the case where cell wall-degrading enzyme was added but alkali metal salt was not. This indicates that the RNA extraction efficiency of S. epidermidis is increased by the addition of cell wall-degrading enzyme. As shown in Figure 3, in the case where cell wall-degrading enzyme was added to S. epidermidis, there was a tendency for the results to be worse (higher Ct value) as the alkali metal salt concentration increased. It was hypothesized that when the alkali metal salt concentration in the reaction solution is high, the cell wall-degrading enzyme denatures or becomes non-functional, so the cell wall is not sufficiently destroyed and the RNA extraction efficiency decreases.

[0050] In cases where cell wall-degrading enzymes were added to C. albicans, although not as pronounced as with S. epidermidis, there was a tendency for performance to worsen (higher Ct values) with increasing alkali metal salt concentrations (Figure 3).

[0051] For bacteria that are difficult to lyse (e.g., S. epidermidis), the use of cell wall-degrading enzymes is desirable. However, the use of cell wall-degrading enzymes rapidly destroys the cells of easily lysable bacteria (e.g., E. coli), leading to RNA degradation. To suppress the rapid cell destruction of easily lysable bacteria, it is desirable to add alkali metal salts to suppress the activity of cell wall-degrading enzymes. However, if the alkali metal salt concentration in the reaction solution is too high, the cell wall-degrading enzymes will denature or become non-functional, reducing the RNA extraction efficiency of bacteria that are difficult to lyse. Therefore, to comprehensively extract RNA from microorganisms, it is necessary to use cell wall-degrading enzymes and adjust the alkali metal ion concentration in the reaction solution to an appropriate range. From the results of Experiment 3, it was shown that the final concentration of alkali metal salts in the reaction solution should be between 50 mM and 150 mM.

[0052] <Experiment 4: Comprehensive RNA extraction from 21 types of bacteria and 3 types of fungi> The 21 types of bacteria and 3 types of fungi shown in Table 5 were cultured. The cultured bacteria were extracted in sterile water in 2 × 10⁻⁶ solutions. ４ The bacterial suspensions of each bacterium were prepared by diluting them to CFU / mL. 500 μL of bacterial suspension (1 × 10⁶) ４ Four types of cell wall-degrading enzymes and alkali metal salts were added to CFU (Sample), and the mixture was incubated at 37°C for 15 minutes to degrade the cell wall.

[0053] • Lysozyme 1 mg / mL • Chitalase 0.5 mg / mL • Glucoamylase 0.5 mg / mL • Achromopeptidase 200 units / mL • Sodium chloride final concentration 100 mM

[0054] Next, the following reagents and proteinase K were added, and the mixture was incubated at 56°C for 20 minutes to decompose the proteins in the reaction solution.

[0055] ・Lysis Buffer 300μL ・Lytic Enhancer 150μL ・Proteinase K 0.675mg / mL

[0056] Next, nucleic acid extraction was performed using the Boom method with silica-coated magnetic beads. DNA hydrolysis with DNase I was carried out during this process. The recovered and washed magnetic beads were suspended in 100 μL of Elution Buffer, and then the magnetic beads were removed to obtain the RNA sample.

[0057] RT-qPCR was performed using RNA samples as templates, targeting conserved bacterial rRNA sequences or conserved fungal rRNA sequences. The results are shown in Table 5.

[0058]

[0059] The highest Ct value was for S. epidermidis, a bacterium that is difficult to lyse, but even then it was 27.1, which is below 30, indicating that a sufficient amount of RNA was extracted from the bacterial cells.

[0060] The results of Experiment 4 demonstrated that by adjusting the alkali metal salt concentration in the reaction solution to an appropriate level and using multiple types of cell wall-degrading enzymes, RNA could be comprehensively extracted from a total of 24 types of bacteria and fungi.

[0061] Based on the experimental results described above, this disclosure provides a novel RNA extraction method. The RNA extraction method and RNA extraction reagent set of this disclosure are described below.

[0062] <RNA Extraction Method> The RNA extraction method described herein is a method for extracting RNA from microorganisms contained in a sample. To extract RNA from microorganisms (typically bacteria and fungi), it is necessary to remove the cell wall. It is preferable to remove the cell wall using cell wall-degrading enzymes. This is because removal of the cell wall using cell wall-degrading enzymes is less likely to cause rapid cell destruction compared to methods of mechanically or chemically removing the cell wall, and therefore can suppress RNA degradation by ribonucleases. However, since different types of microorganisms have different resistance to degradation by cell wall-degrading enzymes, and different mechanical resistance to the osmotic pressure of the cell wall and cell membrane, it is not easy to extract RNA without degradation in a single reaction solution regardless of the type of microorganism.

[0063] Therefore, the RNA extraction method of this disclosure includes the following steps (1), (2-1), and (3) in order to comprehensively extract RNA from microorganisms in a single reaction solution.

[0064] Step (1): Add alkali metal salt and cell wall-degrading enzyme to the sample to prepare a reaction solution containing alkali metal salt and cell wall-degrading enzyme, with a final concentration of alkali metal salt of 50 mM to 150 mM. Step (2-1): Incubate the reaction solution to degrade the cell walls of microorganisms contained in the reaction solution. Step (3): Extract RNA from the reaction solution after incubation.

[0065] The reaction solution prepared in step (1) contains an alkali metal salt and a cell wall-degrading enzyme, and the alkali metal salt concentration is 50 mM to 150 mM. The alkali metal salt concentration of 50 mM to 150 mM in the reaction solution is an appropriate ionic strength for the activity of the cell wall-degrading enzyme to be expressed. When this reaction solution is incubated, if the sample contains microorganisms, the cell walls of the microorganisms are degraded by the cell wall-degrading enzyme (i.e., step (2-1)). Although the type of microorganism contained in the sample is unknown, the alkali metal salt concentration of the reaction solution is in the range of 50 mM to 150 mM, so that any microorganisms are spared from rapid cell destruction and RNA degradation by ribonuclease is suppressed. Then, in step (3), RNA is extracted from the reaction solution after incubation.

[0066] Therefore, according to the RNA extraction method of this disclosure, it is possible to extract RNA from all microorganisms contained in a sample, regardless of the type of microorganism.

[0067] If the alkali metal salt concentration in the reaction solution is less than 50 mM, the cell wall-degrading enzyme functions well, rapidly destroying the cells of microorganisms that have relatively weak resistance to degradation by the cell wall-degrading enzyme, and the RNA is degraded by ribonuclease. From the viewpoint of mitigating the function of the cell wall-degrading enzyme, the alkali metal salt concentration in the reaction solution is 50 mM or higher, preferably 60 mM or higher, more preferably 70 mM or higher, even more preferably 80 mM or higher, and particularly preferably 90 mM or higher. If the alkali metal salt concentration in the reaction solution exceeds 150 mM, the cell wall-degrading enzyme denatures or becomes non-functional, preserving the cells of microorganisms that are difficult to lyse without the action of the cell wall-degrading enzyme, and reducing the RNA extraction efficiency. From the viewpoint of enabling the cell wall-degrading enzyme to function, the alkali metal salt concentration in the reaction solution is 150 mM or lower, preferably 140 mM or lower, more preferably 130 mM or lower, even more preferably 120 mM or lower, and particularly preferably 110 mM or lower.

[0068] The elements and steps of the RNA extraction method described herein will be explained in detail below.

[0069] [Sample] The origin of the sample is not limited. The sample may originate from, for example, an object to be tested for microbiological purposes. Examples of objects to be tested for microbiological purposes include pharmaceuticals, quasi-drugs, cosmetics, active pharmaceutical ingredients, reagents, food, food ingredients, health foods, supplements, animal feed, drinking water, tap water, medical gases, medical components, medical devices, sanitary materials, biological samples, blood, and cultured cells. Examples of pharmaceuticals include small molecule drugs, medium molecule drugs (e.g., peptide drugs, nucleic acid drugs), biopharmaceuticals (e.g., antibody drugs), cell therapies, and gene therapies.

[0070] If the substance to be tested for microbiological examination is a liquid or suspension, a portion of the liquid or suspension is taken as a sample. If the substance to be tested for microbiological examination is a powder, for example, the powder is dissolved or suspended in sterile water to prepare the sample. If the substance to be tested for microbiological examination is a solid, for example, the solid surface is washed with sterile water, and this washing solution is used as a sample. If the substance to be tested for microbiological examination is a gas, for example, the gas is passed through a sterile filter, and then the filter is washed with sterile water, and this washing solution is used as a sample.

[0071] Typical examples of samples are pharmaceuticals. The RNA extraction method of this disclosure is suitable for the purpose of performing microbiological testing on pharmaceuticals with short shelf lives after manufacture (e.g., cell therapies, gene therapies).

[0072] [Microorganisms] Suitable microorganisms from which RNA can be extracted by the RNA extraction method of this disclosure include bacteria and / or fungi.

[0073] Examples of bacteria and fungi include the 21 bacterial species and 3 fungal species listed in Table 5. These are bacteria and fungi listed in ISO 24190:2023 "Biotechnology - Analytical methods - Risk-based approach for method selection and validation for rapid microbial detection in bioprocesses".

[0074] [RNA] The RNA extraction method described herein extracts RNA present in the cells of microorganisms. Examples of RNA to be extracted include mRNA (messenger RNA), rRNA (ribosomal RNA), and tRNA (transfer RNA).

[0075] [Step (1)] Step (1) is a step in which an alkali metal salt and a cell wall-degrading enzyme are added to the sample to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, and in which the final concentration of the alkali metal salt is 50 mM to 150 mM.

[0076] The solvent and dispersion medium of the reaction solution are water. The water may be water derived from the sample, water added when the alkali metal salt and / or cell wall-degrading enzyme is added to the sample, or water added to the sample.

[0077] Examples of alkali metal salts include alkali metal halides. Examples of alkali metal halides include alkali metal fluorides, alkali metal chlorides, alkali metal bromides, and alkali metal iodides. Here, lithium, sodium, and potassium are preferred as alkali metals. There may be one or more alkali metal salts.

[0078] From the viewpoint of alkali metal salts, alkali metal chlorides are preferred, and at least one selected from the group consisting of sodium chloride, potassium chloride, and lithium chloride is more preferred. One type of alkali metal chloride or two or more types may be used.

[0079] The alkali metal salt concentration in the reaction solution is 50 mM to 150 mM. If multiple types of alkali metal salts are present in the reaction solution, the alkali metal salt concentration is the total concentration of those multiple types of alkali metal salts. An alkali metal salt concentration of 50 mM to 150 mM in the reaction solution is an appropriate ionic strength for the activity of cell wall-degrading enzymes.

[0080] The reaction solution has an alkali metal salt concentration of 50 mM to 150 mM, which provides an appropriate osmotic pressure difference with microbial cells. Therefore, the microbial cells contained in the reaction solution are not rapidly destroyed, and as a result, RNA degradation by ribonuclease can be suppressed. Furthermore, the cell membranes of the microbial cells contained in the reaction solution are not excessively preserved, resulting in excellent RNA extraction efficiency. From the above viewpoint, the alkali metal salt concentration of the reaction solution is preferably 60 mM to 140 mM, more preferably 70 mM to 130 mM, even more preferably 80 mM to 120 mM, and particularly preferably 90 mM to 110 mM.

[0081] Cell wall-degrading enzymes are not limited to any type of enzyme that breaks down the cell wall of microorganisms. Examples of cell wall-degrading enzymes include lysozyme, achromopeptidase, glucoamylase, glucanase, labianase, yatarase, zymolyase, and chitinase. Glucanase is an enzyme that primarily exhibits glucanase activity, and glucanase also includes chitarase, among others.

[0082] The cell wall-degrading enzyme may be one type or two or more types. From the viewpoint of comprehensively extracting RNA from microorganisms contained in the sample, it is preferable to use a combination of two to four types of cell wall-degrading enzymes.

[0083] The optimal type of cell wall-degrading enzyme varies depending on the type of microorganism. From the viewpoint of acting on a relatively large number of microorganisms (i.e., from the viewpoint of having a broad lysis spectrum), it is preferable that the cell wall-degrading enzyme contains at least one selected from the group consisting of lysozyme, achromopeptidase, glucoamylase, and glucanase.

[0084] From the viewpoint of comprehensively extracting RNA from microorganisms contained in the sample, it is preferable to use a combination of multiple types of cell wall-degrading enzymes, each with different substrates. An example of a preferred combination is a combination of lysozyme, achromopeptidase, and at least one selected from the group consisting of glucoamylase and glucanase. Specific examples of preferred combinations include a combination of three types: lysozyme, achromopeptidase, and glucoamylase; a combination of three types: lysozyme, achromopeptidase, and glucanase; and a combination of four types: lysozyme, achromopeptidase, glucoamylase, and glucanase.

[0085] The concentration of cell wall-degrading enzymes in the reaction solution should be the recommended amount for each type of enzyme. The concentration of lysozyme should be 0.75 mg / mL to 2 mg / mL. The concentration of achromopeptidase should be 50 U / mL to 2000 U / mL. The concentration of glucoamylase should be 0.25 g / mL to 2 g / mL. The concentration of glucanase should be 0.25 g / mL to 2 g / mL.

[0086] The reaction solution contains, in addition to microorganisms, at least alkali metal salts and cell wall-degrading enzymes, and may also contain other components. Examples of other components include nutrients for microorganisms (e.g., peptone, glucose), stabilizers for each component, pH adjusters, and pH buffers. The pH of the reaction solution is preferably in the range of 6.0 to 8.0.

[0087] An example of an embodiment of process (1) includes the following steps (1a) and (1b).

[0088] Step (1a): Add an alkali metal salt to the sample and pre-incubate it. Step (1b): Add a cell wall-degrading enzyme to the sample after pre-incubation to prepare a reaction solution containing an alkali metal salt and a cell wall-degrading enzyme, with a final concentration of alkali metal salt of 50 mM to 150 mM.

[0089] Step (1a) activates the activity of microorganisms, increasing the amount of RNA within the microbial cells, which in turn can lead to an expected increase in the amount of RNA extracted. From the viewpoint of further activating microbial activity, it is preferable to add a nutrient source for microorganisms (e.g., peptone, glucose) to the sample in step (1a).

[0090] Step (1a) is carried out, for example, by allowing the sample to be given an alkali metal salt to stand or be stirred at a temperature of 37°C for 30 to 120 minutes.

[0091] [Step (2-1)] Step (2-1) is a step in which the reaction solution is incubated and the cell walls of microorganisms contained in the reaction solution are broken down.

[0092] Step (2-1) is achieved by maintaining the reaction mixture at a temperature at which the cell wall-degrading enzymes function for a certain period of time. For example, the reaction mixture is left to stand or stirred at 37°C for 10 to 30 minutes.

[0093] [Step (2-2)] An example of an embodiment of the RNA extraction method of the present disclosure further includes the following step (2-2) between step (2-1) and step (3).

[0094] Step (2-2): Add a proteolytic enzyme to the reaction solution after incubation to break down the proteins contained in the reaction solution.

[0095] Proteins degraded by step (2-2) include enzymes (e.g., cell wall degrading enzymes, ribonucleases), RNA-bound proteins (e.g., ribosomal proteins), microbial cell wall proteins, microbial cell membrane proteins, and contaminants derived from the sample. By performing step (2-2), an increase in the amount of RNA extracted can be expected.

[0096] Examples of proteases include proteases. Examples of proteases include serine proteases (e.g., proteinase K, trypsin), cysteine ​​proteases (papain), threonine proteases, aspartate proteases (e.g., pepsin), glutamate proteases, metalloproteases, and asparagine peptide lyases. One type of protease may be used, or two or more types may be used.

[0097] In step (2-2), proteinase K is preferred as the proteolytic enzyme used, from the viewpoint of rapidly degrading ribonuclease and having a wide pH and temperature range in which it is active.

[0098] The concentration of proteolytic enzymes in the reaction solution should be the recommended amount for each type of enzyme. The concentration of serine protease should be 0.5 mg / mL to 2 mg / mL. The concentration of proteinase K should be 0.5 mg / mL to 2 mg / mL.

[0099] In step (2-2), it is preferable to add a chaotropic salt and / or a surfactant to the reaction solution, and more preferably to add both a chaotropic salt and a surfactant to the reaction solution.

[0100] Examples of chaotropic salts include sodium iodide, potassium iodide, sodium bromide, potassium bromide, calcium bromide, ammonium bromide, urea, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, guanidine hydrochloride, and sodium thiocyanate. Among these, sodium iodide and potassium iodide are preferred. Examples of surfactants include cationic surfactants such as cetyltrimethylammonium bromide; anionic surfactants such as sodium dodecyl sulfate, sodium cholate, and sodium N-dodecanoylsarcosinate; amphoteric surfactants such as CHAPS and CHAPSO; and nonionic surfactants such as Triton X-100, Tween 20, Tween 40, Tween 60, and Tween 80. Among these, anionic surfactants are preferred, and specifically, sodium dodecyl sulfate and sodium N-dodecanoylsarcosinate are preferred. Commercially available cell lysis reagents or cell lysis enhancers may be used as the chaotropic salt and / or surfactant.

[0101] The decomposition of proteins in the reaction solution is achieved by adding a proteolytic enzyme to the reaction solution and then maintaining the reaction solution at a temperature at which the proteolytic enzyme functions for a certain period of time. For example, the reaction solution is left to stand or stirred at a temperature of 37°C to 60°C for 10 to 60 minutes.

[0102] [Step (3)] Step (3) is the step of extracting RNA from the reaction mixture after incubation. The method of extracting RNA from the reaction mixture is not limited, and any known nucleic acid extraction method can be applied.

[0103] During the process of extracting RNA from the reaction solution, it is preferable to hydrolyze the DNA using deoxyribonuclease. Hydrolyzing the DNA suppresses the contamination of the extract with DNA.

[0104] As the deoxyribonuclease used in step (3), DNaseI is preferred from the viewpoint of being able to hydrolyze both single-stranded and double-stranded DNA.

[0105] As an example of an embodiment of process (3), the Boom method can be cited. An example of an embodiment of the Boom method is described below. Any known process and operation can be applied to the series of processes and operations described below.

[0106] A protease, chaotropic salt, and surfactant are added to the reaction mixture to homogenize it (i.e., step (2-2) is performed before step (3)). Silica-coated magnetic beads are added to the homogenized reaction mixture to allow nucleic acids to adhere to the silica on the surface of the magnetic beads. Next, deoxyribonuclease (preferably DNaseI) is added to hydrolyze the DNA and remove it from the surface of the magnetic beads. The magnetic beads are recovered by magnetism and washed. RNA is separated from the washed magnetic beads.

[0107] It is preferable to purify the RNA extracted from the reaction solution by washing and concentrating it. The purified RNA should be stored refrigerated or frozen.

[0108] RNA extracted by the RNA extraction method disclosed herein can be subjected to microbiological testing, for example, by nucleic acid amplification testing (NAT). RNA-targeted NAT includes reverse transcription and PCR. Reverse transcription and PCR use primers specific to the RNA sequence to be detected.

[0109] <RNA Extraction Reagent Set> The RNA extraction reagent set of this disclosure is a reagent set for extracting RNA from microorganisms contained in a sample, and is a reagent set for carrying out the RNA extraction method of this disclosure.

[0110] The samples, microorganisms, and RNA morphologies targeted by the RNA extraction reagent set of this disclosure are as previously described in the description of the RNA extraction method of this disclosure.

[0111] The RNA extraction reagent set disclosed herein comprises at least an alkali metal salt solution having an alkali metal salt concentration of 50 mM or higher, and a cell wall-degrading enzyme. The alkali metal salt solution and the cell wall-degrading enzyme are housed in separate containers. A set of reagents housed in these separate containers constitutes the reagent set.

[0112] The RNA extraction reagent set disclosed herein makes it possible to comprehensively extract RNA from microorganisms contained in a sample, regardless of the type of microorganism.

[0113] [Alkali metal salt solution] The alkali metal salt solution is used in step (1) of the RNA extraction method of this disclosure. The alkali metal salt solution is added to the sample to prepare a reaction solution with an alkali metal salt concentration of 50 mM to 150 mM.

[0114] The alkali metal salt solution contains at least an alkali metal salt in the solvent. The type of alkali metal salt is as described above in the explanation of step (1), and the preferred type is also as described above. Water is preferred as the solvent for the alkali metal salt solution.

[0115] When adding the alkali metal salt solution to the sample in step (1), the amount added is adjusted to prepare the alkali metal salt concentration of the reaction solution to 50 mM to 150 mM. The alkali metal salt concentration of the alkali metal salt solution is 50 mM or higher and below the saturation concentration. From the viewpoint of solution stability at room temperature (15°C to 25°C), the alkali metal salt concentration of the alkali metal salt solution is preferably 3 M or lower.

[0116] From the viewpoint of minimizing the volume of the reaction solution prepared in step (1), the alkali metal salt concentration of the alkali metal salt solution is preferably greater than 50 mM to 3 M, preferably 75 mM to 3 M, more preferably 90 mM to 3 M, even more preferably 100 mM to 3 M, even more preferably 110 mM to 3 M, and particularly preferably 120 mM to 3 M.

[0117] Alkali metal salt solutions may contain other components besides alkali metal salts. Examples of other components include nutrients for microorganisms (e.g., peptone, glucose), pH adjusters, and pH buffers.

[0118] [Cell wall-degrading enzyme] The cell wall-degrading enzyme is used in step (1) of the RNA extraction method of this disclosure. The cell wall-degrading enzyme is added to the sample to prepare the reaction solution.

[0119] The types of cell wall-degrading enzymes are as described above in the explanation of step (1), and the preferred types and combinations are also as described above.

[0120] If there are multiple types of cell wall-degrading enzymes, they may be housed in separate containers for each type of enzyme, or all enzymes may be housed in a single container. The cell wall-degrading enzymes are housed in containers, for example, dissolved or dispersed in 50% (v / v) glycerol / buffer solution.

[0121] [Protease] An example of an embodiment of the RNA extraction reagent set of this disclosure further includes a protease. The protease is used in step (2-2) of the RNA extraction method of this disclosure. The type of protease is as described above in the description of step (2-2), and the preferred type is also as described above.

[0122] The proteolytic enzymes are housed in separate containers from the alkali metal salt solutions and cell wall-degrading enzymes. For example, the proteolytic enzymes are housed in containers dissolved or dispersed in 50% (v / v) glycerol / buffer solution.

[0123] [Chaotropic Salts and / or Surfactants] An example of an embodiment of the RNA extraction reagent set of the present disclosure further comprises chaotropic salts and / or surfactants. The chaotropic salts and / or surfactants are used in step (2-2) of the RNA extraction method of the present disclosure. The types of chaotropic salts and surfactants are as described above in the description of step (2-2).

[0124] The chaotropic salt and / or surfactant is housed in a separate container from the alkali metal salt solution and enzyme. The RNA extraction reagent set of this disclosure may include known cell lysis reagents or cell lysis enhancers as the chaotropic salt and / or surfactant.

[0125] [Deoxyribonuclease] An example of an embodiment of the RNA extraction reagent set of the present disclosure further comprises a deoxyribonuclease. The deoxyribonuclease is used in step (3) of the RNA extraction method of the present disclosure. The type of deoxyribonuclease is as described above in the description of step (3), and the preferred type is also as described above.

[0126] Deoxyribonuclease is contained in a separate container from alkali metal salt solutions and other enzymes. Deoxyribonuclease is contained in a container in a form dissolved or dispersed in, for example, 50% (v / v) glycerol / buffer solution.

[0127] [Reagents for the Boom Method] An example embodiment of the RNA extraction reagent set of the present disclosure further includes reagents for the Boom method (e.g., silica-coated magnetic beads, washing buffer, elution buffer). The reagents for the Boom method are used in step (3) of the RNA extraction method of the present disclosure.

[0128] The RNA extraction reagent set disclosed herein may include equipment used for RNA extraction.

[0129] The RNA extraction reagent set of this disclosure is preferably distributed in a form that preserves enzyme activity, for example, through low-temperature storage.

[0130] The RNA extraction method of this disclosure will be explained below with reference to examples. The following examples illustrate the application of the RNA extraction method of this disclosure to rapid microbiological testing of pharmaceuticals. In the following examples, the RNA extracted by the RNA extraction method of this disclosure is subjected to RT-qPCR. The materials, amounts used, concentrations, processing procedures, etc., shown in the following examples can be modified as appropriate without departing from the spirit of this disclosure. Therefore, the scope of the RNA extraction method of this disclosure should not be interpreted as being limited by the specific examples shown below.

[0131] <Rapid Microbiological Testing of Cell Therapies> [Preparation of Primer Sets and Probes] Prepare a bacterial-specific primer set, a bacterial-specific fluorescent dye-labeled probe, a fungal-specific primer set, and a fungal-specific fluorescent dye-labeled probe for use in RT-qPCR. The above primer sets and probes may be commercially available products or newly designed and manufactured primer sets and probes. An example of a method for designing primer sets and probes is shown below.

[0132] Based on the rRNA sequence database information for 21 bacterial species shown in Table 6, highly homologous conserved sequences will be identified among the 21 bacterial species. A PCR primer set and probes will be designed to amplify and detect these conserved sequences. The reverse primers will also function as reverse transcription primers. Based on the rRNA sequence database information for 3 fungal species shown in Table 6, highly homologous conserved sequences will be identified among the 3 fungal species. A PCR primer set and probes will be designed to amplify and detect these conserved sequences. The reverse primers will also function as reverse transcription primers.

[0133] The bacteria and fungi listed in Table 6 are microorganisms listed in ISO 24190:2023 "Biotechnology - Analytical methods - Risk-based approach for method selection and validation for rapid microbial detection in bioprocesses".

[0134]

[0135] Bacteria-specific probes and fungal-specific probes are labeled with fluorescent dyes for fluorescence monitoring. Preferably, the fluorescent dyes of the bacteria-specific probe and the fungal-specific probe have the same principle of controlling fluorescence emission. The fluorescent dyes of the bacteria-specific probe and the fungal-specific probe may be the same or different. If the fluorescent dyes are different, bacteria and fungi can be detected separately.

[0136] [Sample Preparation] Cell preparations are autologous cell preparations for cancer treatment, manufactured, for example, by introducing specific genes into T cells collected from the blood of cancer patients.

[0137] A portion of the cell preparation is collected, and the cells are separated by centrifugation, or by filtration using a filter that allows microorganisms to pass through. After separating the cells, ultracentrifugation may be performed to concentrate the microorganisms. The recovered microorganisms are suspended in a buffer solution to obtain a suspension.

[0138] [RNA Extraction (RNA Extraction Method of the Present Disclosure)] To the above suspension, an alkali metal salt solution with an alkali metal salt concentration of 150 mM and a cell wall-degrading enzyme are added to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, with a final alkali metal salt concentration of 100 mM. ...Step (1)

[0139] The reaction solution is incubated to break down the cell walls of microorganisms contained in the solution. ...Step (2-1)

[0140] After incubation, a proteolytic enzyme, chaotropic salt, and surfactant are added to the reaction mixture to homogenize it. ...Step (2-2)

[0141] Silica-coated magnetic beads are added to the homogenized reaction solution, and nucleic acids are attached to the silica on the surface of the magnetic beads. Next, DNaseI is added to hydrolyze the DNA and remove it from the surface of the magnetic beads. The magnetic beads are recovered by magnetism and washed. RNA is separated from the washed magnetic beads. The separated RNA is washed and concentrated to obtain an RNA sample. ...Step (3)

[0142] [RT-qPCR (Multiplex RT-qPCR)] An RT-qPCR reaction solution is prepared by adding a bacterial-specific primer set, a bacterial-specific fluorescent dye-labeled probe, a fungal-specific primer set, a fungal-specific fluorescent dye-labeled probe, dNTPs, reverse transcriptase, and DNA polymerase to an RNA sample. RT-qPCR is then performed using this RT-qPCR reaction solution.

[0143] Real-time quantification is performed using a fluorescence monitoring method. PCR is repeated a predetermined number of times while monitoring the fluorescence intensity. When the fluorescence intensity reaches the threshold, it is determined to be positive (i.e., the cell preparation is contaminated with bacteria and / or fungi). When the fluorescence intensity does not reach the threshold, it is determined to be negative (i.e., the cell preparation is not contaminated with bacteria and / or fungi). The PCR threshold and the number of cycles are predetermined by preliminary experiments.

[0144] By automating sample preparation, RNA extraction, and RT-qPCR, it is possible to perform rapid microbiological testing of cell preparations in a sterile room without human intervention.

[0145] All documents, patent applications, and technical standards described herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

[0146] The disclosure of Japanese application number 2024-171186, filed on 30 September 2024, is incorporated herein by reference in its entirety.

Claims

1. A method for extracting RNA from microorganisms contained in a sample, comprising the following steps: (1) adding an alkali metal salt and a cell wall-degrading enzyme to the sample to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, wherein the final concentration of the alkali metal salt is 50 mM to 150 mM; (2) incubating the reaction solution to degrade the cell walls of microorganisms contained in the reaction solution; and (3) extracting RNA from the reaction solution after incubation.

2. The RNA extraction method according to claim 1, further comprising (2-2) below between (2-1) and (3); (2-2) adding a proteolytic enzyme to the reaction solution after incubation to decompose the proteins contained in the reaction solution.

3. The RNA extraction method according to claim 1, wherein (3) above includes degrading DNA using a deoxyribonuclease.

4. The RNA extraction method according to claim 1, wherein (1) comprises the following (1a) and (1b): (1a) adding an alkali metal salt to a sample and pre-incubating it; (1b) adding a cell wall-degrading enzyme to the sample after pre-incubation to prepare a reaction solution containing the alkali metal salt and the cell wall-degrading enzyme, wherein the final concentration of the alkali metal salt is 50 mM to 150 mM.

5. The RNA extraction method according to any one of claims 1 to 4, wherein the alkali metal salt comprises at least one selected from the group consisting of sodium chloride, potassium chloride, and lithium chloride.

6. The RNA extraction method according to any one of claims 1 to 4, wherein the cell wall degrading enzyme comprises at least one selected from the group consisting of lysozyme, achromopeptidase, glucoamylase, and glucanase.

7. The RNA extraction method according to any one of claims 1 to 4, wherein the cell wall degrading enzyme comprises lysozyme, achromopeptidase, and at least one selected from the group consisting of glucoamylase and glucanase.

8. The RNA extraction method according to claim 2, wherein the proteolytic enzyme comprises proteinase K.

9. The RNA extraction method according to any one of claims 1 to 4, wherein the microorganism is a bacterium and / or a fungus.

10. A reagent set for extracting RNA from microorganisms contained in a sample, comprising an alkali metal salt solution with an alkali metal salt concentration of 50 mM or higher, and a cell wall-degrading enzyme, for RNA extraction.

11. The RNA extraction reagent set according to claim 10, further comprising a protease and / or deoxyribonuclease.

12. The RNA extraction reagent set according to claim 10 or claim 11, wherein the alkali metal salt comprises at least one selected from the group consisting of sodium chloride, potassium chloride, and lithium chloride.

13. The RNA extraction reagent set according to claim 10 or claim 11, wherein the cell wall degrading enzyme comprises at least one selected from the group consisting of lysozyme, achromopeptidase, glucoamylase, and glucanase.

14. The RNA extraction reagent set according to claim 10 or claim 11, wherein the cell wall degrading enzyme comprises lysozyme, achromopeptidase, and at least one selected from the group consisting of glucoamylase and glucanase.

15. The RNA extraction reagent set according to claim 11, wherein the proteolytic enzyme comprises proteinase K.

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