Blood cell lysis agents for isolating bacteria from blood cultures

Abstract

Disclosed are methods, compositions, and kits suitable for processing a sample containing blood cells and at least one microorganism. In some embodiments, the method includes contacting the sample with a lysis buffer to produce a processed sample. The lysis buffer can include a somatic cell digestion agent (SDA) capable of lysing blood cells in the sample. In some embodiments, the at least one microorganism remains intact and / or viable in the presence of the SDA.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 068,278, filed on August 20, 2020, pursuant to Section 119(e) of the U.S. Patent Act. The entire contents of these applications are expressly incorporated herein by reference throughout them. [Background technology]

[0002] background field This disclosure relates in general to the field of microbial isolation and identification.

[0003] Explanation of related technologies Sepsis is a serious medical condition resulting from a severe reaction of the host's immune system to infection. It can cause widespread inflammation and impaired blood flow. As sepsis progresses, the body's organs can become deprived of oxygen and nutrients, leading to permanent damage and eventual failure. If left undiagnosed or even untreated, the heart can fail, leading to septic shock, multiple organ failure, and death. Blood cultures are necessary to detect the presence of bacteria or yeast in the blood of patients with sepsis. If microorganisms are present (positive blood culture ("PBC")), the microorganisms must be identified and their antibiotic susceptibility determined in order to provide appropriate treatment. PBC samples are used for isolation, identification, and antimicrobial susceptibility testing ("AST"). Microorganisms are often identified by mass spectrometry or phenotypic growth-based methods, including MALDI-TOF / MS, such as Phoenix® ID.

[0004] To identify microorganisms, perform phenotypic analysis on them, and conduct AST testing, it is necessary to isolate intact and / or viable microorganisms from blood cells and other materials in the collected sample. For microbial identification by mass spectrometry, substances known to interfere with MALDI-TOF / MS identification, such as blood cell components, other cell debris, and salts, must be thoroughly removed from the microbial sample. Furthermore, the microbial sample must be of sufficient quantity to achieve reliable identification. Phenotypic identification methods, such as Phoenix® ID, require intact and viable microorganisms free from substances that may interfere with the assay's enzyme substrates. For AST tests such as Phoenix® AST, the microbial sample must contain viable, unchanging microorganisms that can grow in the presence of antibiotics during the assay, if resistance mechanisms exist. For all methods, it is important that residual blood or culture medium components are present in sufficient quantity and purity, as carryover will interfere directly or unduly by increasing the microbial concentration (turbidity).

[0005] Current techniques for isolating viable microorganisms from PBC samples involve microbial subculturing, which can take up to 72 hours. This can lead to delayed treatment or treatment with inappropriate antibiotics. For example, isolating specific strains of microorganisms from PBC samples while maintaining the viability of organisms such as Streptococcus pneumoniae (S. pneumoniae) is particularly difficult. Part of this difficulty stems from the activation of autolytic enzymes by Streptococcus pneumoniae, which causes the microbial cells to "self-destruct." See “Streptococcus pneumoniae Antigen Test Using Positive Blood Culture Bottles as an Alternative Method To Diagnose Pneumococcal Bacteremia”, Journal of Clinical Microbiology, Vol. 43, No. 5, May 2005, pp. 2510-2512. Current methods for isolating microorganisms, including Streptococcus pneumoniae, from sepsis patients involve inoculation of blood culture bottles. Once a positive signal is achieved, a portion of the PBC sample is taken and Gram-stained, and another portion is used to passage the microorganism. Downstream tests are performed using microbial colonies from successive generations, such as identification by MALDI-TOF / MS, phenotypic identification, and AST testing.

[0006] Further techniques for isolating viable microorganisms from PBC samples often involve liquid separation methods that include a lysis buffer containing a surfactant to lyse the blood cells in the PBC sample. After lysis, the lysed blood cells can be removed while retaining the microorganisms. However, the use of these lysis buffers often results in impaired, damaged, or infertile microorganisms, making them insufficient for specific growth-based identification methods such as AST testing. Currently available sample preparation methods and compositions suffer from various shortcomings. For example, (i) insufficient viability due to the interaction of coarse surfactants on the microbial cell wall after sample preparation to support growth-based identification and AST methods; (ii) resulting in inconsistent identification of microorganisms at the seed level across a panel of microorganisms; and / or (iii) inability to isolate viable microorganisms from a PBC sample that do not contain interfering substances and enable multiple downstream tests from a single PBC sample, such as both MALDI-TOF / MS identification and AST testing. Therefore, efficient hematopoietic agents are needed to isolate microorganisms from positive blood cultures for rapid MALDI identification. Hematopoietic agents are also needed to identify difficult bacterial species, such as Staphylococcus epidermidis, which yield low MALDI scores when isolated using currently available methods from positive blood cultures. [Overview of the project]

[0007] overview This specification discloses a method for processing a sample. The method involves contacting a sample containing blood cells and at least one microorganism with a lysis buffer to produce a processed sample, wherein the lysis buffer contains a somatic cell digestion agent (SDA) capable of lysing the blood cells in the sample, and the SDA is expressed by the following formula 1

[0008] [ka]

[0009] A compound (where x is an integer from 2 to 20 and y is an integer from 6 to 11), and a step of lysing blood cells in a sample thereby. In some embodiments, y is an integer from 8 to 10. In some embodiments, y is 8. In some embodiments, x is an integer from 5 to 15. In some embodiments, x is an integer from 8 to 12, such as 9 or 10. In some embodiments, x is 9. In some embodiments, SDA is nonoxynol-9. In some embodiments, the concentration of SDA in the lysis buffer is from about 0.01 g / L to about 10 g / L. In some embodiments, the concentration of SDA in the lysis buffer is from about 0.01% (w / w) to about 10% (w / w). In some embodiments, the concentration of SDA in the lysis buffer is from about 0.01% (w / w) to about 1% (w / w). In some embodiments, the concentration of SDA in the lysis buffer is about 0.52% (w / w). In some embodiments, the sample is derived from a blood culture of a subject suspected of being infected. In some embodiments, the sample includes a positive blood culture sample determined to contain at least one microorganism therein. In some embodiments, the at least one microorganism is selected from the group including gram-positive bacteria, gram-negative bacteria, and yeast. In some embodiments, the at least one microorganism is Staphylococcus epidermidis (S. epidermidis). In some embodiments, the at least one microorganism includes one or more of Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli (E. coli), and Streptococcus pneumoniae (S. pneumoniae).

[0010] In some embodiments, the contact step includes sonication, osmotic shock, chemical treatment, or any combination thereof. In some embodiments, the lysis buffer includes one or more proteinases and / or one or more nucleases. The method may include a step of isolating at least one microorganism from the treated sample to produce at least one isolated microorganism. In some embodiments, the step of isolating at least one microorganism from the treated sample includes a step of separating at least one microorganism from lysed hematopoiesis. In some embodiments, the step of separating at least one microorganism from lysed hematopoiesis includes a step of centrifugation of the treated sample to produce a pellet and a supernatant, and a step of discarding the supernatant while retaining the pellet containing at least one isolated microorganism. The method may include a step of preparing a plated pure culture from at least one isolated microorganism and analyzing the microorganism obtained from this plated pure culture. The method may include a step of preparing an inoculum from at least one isolated microorganism and analyzing at least one microorganism obtained from this inoculum.

[0011] The method can include depositing at least a portion of a pellet containing at least one isolated microorganism onto a surface suitable for placement within an apparatus configured to determine the identity of at least one microorganism by mass spectrometry, optionally drying the deposited sample, treating the deposited sample with a volatile acid solution, where the volume percent of the volatile acid is at least 70% of the volatile acid solution combined with the deposited sample, optionally drying the treated deposited sample, placing a matrix over the treated deposited sample, and optionally drying the treated deposited sample. In some embodiments, the volatile acid solution is an aqueous solution of a volatile acid or a volatile solution in an organic solvent. In some embodiments, the volatile acid solution is an aqueous solution of formic acid at a volume percent of 70% when combined with the deposited sample. In some embodiments, the volatile acid solution is an aqueous solution of formic acid at a volume percent of 80% when combined with the deposited sample. In some embodiments, the volatile acid solution is an aqueous solution of formic acid at a volume percent of 90% when combined with the deposited sample. The method can include treating the deposited sample with an organic solvent and drying the deposited sample before treating the deposited sample with the volatile acid solution. In some embodiments, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, acetone, ethyl acetate, or any combination thereof.

[0012] The method can include contacting the sample with a choline-containing solution before, simultaneously with, and / or after contact of the sample with the lysis buffer. In some embodiments, the choline-containing solution includes at least one quaternary ammonium salt containing a N,N,N-trimethylethanolammonium cation selected from Formula 2 below.

[0013] [Chemical formula]

[0014] where R 1 、R 2 、and R 3X independently represents a group selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and any combination thereof, and X represents a loaded electrolytic group. In some embodiments, X represents chloride, fluoride, nitric acid, and bicarbonate. In some embodiments, the choline-containing solution contains choline chloride. In some embodiments, the choline-containing solution contains phosphorylcholine. In some embodiments, the final concentration of choline upon contact with the sample is about 0.25% by volume or more. In some embodiments, the final concentration of choline upon contact with the sample is about 1% by volume or more. In some embodiments, the concentration of choline in the sample during the contact step is about 1.8% by volume. In some embodiments, the concentration of choline in the sample during the contact step is about 4% by volume. In some embodiments, the concentration of choline in the sample during the contact step is in the range of about 0.25% by volume to about 10% by volume. In some embodiments, the contact step comprises incubating the sample with the choline-containing solution for up to 20 minutes, the incubation temperature being room temperature.

[0015] The lysis buffer may further contain an antifoaming agent. In some embodiments, the lysis buffer does not contain an antifoaming agent. In some embodiments, the lysis buffer further contains at least one thiol. In some embodiments, the at least one thiol includes L-cysteine ​​HCl, sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid, mercaptoethanol, mercaptoethanesulfonic acid, thioglycerol, or any combination thereof, and the concentration of at least one thiol in the lysis buffer is possibly about 0.005 g / L to 4 g / L. In some embodiments, the at least one thiol includes L-cysteine ​​at a concentration of about 0.01 g / L to about 2.5 g / L in the lysis buffer, and / or sodium thioglycolate at a concentration of about 0.01 g / L to about 2.5 g / L in the lysis buffer. In some embodiments, the lysis buffer further contains ammonium chloride, and the concentration of ammonium chloride in the lysis buffer is approximately 0.01 g / L to about 80 g / L. In some embodiments, the lysis buffer further comprises a nutrient base solution containing one or more of the following in the lysis buffer: casein peptone at a concentration of about 8 g / L to about 35 g / L, sodium chloride at a concentration of about 2 g / L to about 10 g / L, soy peptone at a concentration of about 1.5 g / L to about 15 g / L, potassium phosphate at a concentration of about 0.5 g / L to about 5 g / L, and at least one other nutrient. In some embodiments, the at least one other nutrient comprises a nutrient medium at a concentration of about 10 g / L to about 50 g / L in the lysis buffer. In some embodiments, the at least one other nutrient comprises a nutrient medium containing one or more of the following: i) tryptone; ii) soybean (soy); iii) NaCl; iv) dipotassium phosphate (K2HPO4); and v) glucose.

[0016] In some embodiments, the lysis buffer further comprises one or more of a nutrient medium, isotonic buffer, peptone, and salt, and optionally the concentration of the nutrient medium in the lysis buffer is about 10 g / L to about 50 g / L. In some embodiments, the nutrient medium comprises Tripticase soy medium. In some embodiments, the isotonic buffer comprises sodium phosphate, potassium phosphate, phosphate-buffered saline, physiological saline, or any combination thereof, and optionally the concentration of the isotonic buffer in the lysis buffer is about 1 g / L to about 20 g / L. In some embodiments, the peptone comprises casein peptone and / or soy peptone. In some embodiments, the lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate-buffered saline, or any combination thereof. In some embodiments, the lysis buffer further comprises at least one additional nonionic surfactant, and optionally the at least one additional nonionic surfactant comprises saponin. In some embodiments, the lysis buffer does not contain additional nonionic surfactants.

[0017] The method may include a step of identifying at least one microorganism. In some embodiments, the identification of at least one microorganism may include mass spectrometry, phenotypic identification, antimicrobial susceptibility testing, molecular testing, or a combination thereof. In some embodiments, mass spectrometry may be electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, or ESI-MS / (MS). nMatrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCJ-MS / MS, APCI-(MS) n , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS) n The mass spectrometry includes one or more of the following: quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero. In some embodiments, the mass spectrometry includes MALDI-TOF-MS.

[0018] In some embodiments, SDA does not damage at least one microorganism. For example, at least one microorganism remains intact and / or viable in the presence of SDA. In some embodiments, the method yields a MALDI score at least 5% higher than a comparative method using a lysis buffer without SDA. In some embodiments, the comparative method uses a lysis buffer containing saponin. In some embodiments, the lysis buffer selectively lyses at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the blood cells in the sample. In some embodiments, the ratio of lysed blood cells to lysed cells of at least one microorganism after the contact step is at least about 2:1. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the cells of at least one microbial species remain intact and / or viable after the contact step.

[0019] In some embodiments, the lysis buffer does not contain a buffer. In some embodiments, the lysis buffer is acidic. In some embodiments, the identification of at least one microorganism does not involve spectroscopic measurements, such as intrinsic fluorescence spectroscopy. In some embodiments, the method does not involve density gradient centrifugation. In some embodiments, the lysis buffer does not contain saponins. In some embodiments, the lysis buffer is Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve(trademark) 200, Brij(registered trademark) 96 / 97, CHAPS, Octyl β-D-Glucopyranoside, Saponin, Nonaethylene glycol monododecyl ether (C12E9, polidocenol), Sodium dodecyl sulfate, N-Lauryl sarcosine, Sodium deoxycholate, Bile salt, Hexadecyltrimethylammonium bromide, SB3-10, SB3-12, Amidosulfobetaine-14, C7BzO, Brij(registered trademark) 98, Brij(registered trademark) 58, Brij(registered trademark) 35, Tween(registered trademark) 80, Tween(registered trademark) 20, Pluronic(registered trademark) L64, Pluronic(registered trademark) P84, Non-surfactant sulfobetaine (NDSB The lysis buffer does not contain one or more surfactants selected from the group consisting of 201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, and nonaethylene glycol monododecyl ether.In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of sodium dodecyl sulfate, N-lauroyl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and policosanol. In some embodiments, the lysis buffer has Structure C. 12-18 / E 9-10 (where C 12-18 represents a carbon chain length of 12 to 18 carbon atoms and E 9-10 represents a polyoxyethylene hydrophilic head group of 9 to 10 oxyethylene groups) and does not contain a polyoxyethylene surfactant.

[0020] Disclosed herein are compositions (e.g., kits). In some embodiments, the composition is a lysis buffer containing a somatic cell digestion agent (SDA) capable of lysing blood cells, wherein the SDA has the following formula 1

[0021]

Chemical formula

[0022] (where x is an integer from 2 to 20 and y is an integer from 6 to 11) and contains a lysis buffer, which is a compound of, blood cells and / or their debris. In some embodiments, y is an integer from 8 to 10. In some embodiments, y is 8. In some embodiments, x is an integer from 5 to 15. In some embodiments, x is an integer from 8 to 12. In some embodiments, x is 9 or 10. In some embodiments, x is 9. In some embodiments, the SDA is nonoxynol-9. In some embodiments, the concentration of SDA in the lysis buffer is about 0.01 g / L to about 10 g / L. In some embodiments, the concentration of SDA in the lysis buffer is about 0.01% (w / w) to about 10% (w / w), for example, about 0.01% (w / w) to about 1% (w / w). In some embodiments, the concentration of SDA in the lysis buffer is about 0.52% (w / w). In some embodiments, the lysis buffer contains one or more proteinases and / or one or more nucleases. In some embodiments, the composition is given by the following formula 2

[0023] [ka]

[0024] (In the formula, R 1 , R 2 , and R 3 (where represents a group independently selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and X represents a loaded electrolytic group.) The solution comprises a choline-containing solution comprising at least one quaternary ammonium salt containing an N,N,N-trimethylethanolammonium cation, selected from the group consisting of the following: In some embodiments, X is selected from the group consisting of chloride, fluoride, nitric acid, and bicarbonate. In some embodiments, the choline-containing solution comprises choline chloride. In some embodiments, the choline-containing solution comprises phosphorylcholine.

[0025] In some embodiments, the lysis buffer further contains an antifoaming agent. In some embodiments, the lysis buffer does not contain an antifoaming agent. In some embodiments, the lysis buffer further contains at least one thiol. In some embodiments, the at least one thiol includes L-cysteine ​​HCl, sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid, mercaptoethanol, mercaptoethanesulfonic acid, thioglycerol, or any combination thereof, and optionally, the concentration of the at least one thiol in the lysis buffer is about 0.005 g / L to 4 g / L. In some embodiments, the at least one thiol includes L-cysteine ​​at a concentration of about 0.01 g / L to about 2.5 g / L in the lysis buffer, and / or sodium thioglycolate at a concentration of about 0.01 g / L to about 2.5 g / L in the lysis buffer. In some embodiments, the lysis buffer further contains ammonium chloride, and the concentration of ammonium chloride in the lysis buffer is about 0.01 g / L to about 80 g / L. In some embodiments, the lysis buffer further comprises a nutrient base solution containing one or more of the following in the lysis buffer: casein peptone at a concentration of about 8 g / L to about 35 g / L, sodium chloride at a concentration of about 2 g / L to about 10 g / L, soy peptone at a concentration of about 1.5 g / L to about 15 g / L, and potassium phosphate at a concentration of about 0.5 g / L to about 5 g / L, along with at least one other nutrient. In some embodiments, the at least one other nutrient comprises a nutrient medium at a concentration of about 10 g / L to about 50 g / L in the lysis buffer. In some embodiments, the at least one other nutrient comprises a nutrient medium containing one or more of the following: i) tryptone, ii) soy, iii) NaCl, iv) dipotassium phosphate (K2HPO4), and v) glucose.

[0026] In some embodiments, the lysis buffer further comprises one or more of a nutrient medium, isotonic buffer, peptone, and salt, and optionally the concentration of the nutrient medium in the lysis buffer is about 10 g / L to about 50 g / L. In some embodiments, the nutrient medium comprises Tripticase soy medium. In some embodiments, the isotonic buffer comprises sodium phosphate, potassium phosphate, phosphate-buffered saline, physiological saline, or any combination thereof, and optionally the concentration of the isotonic buffer in the lysis buffer is about 1 g / L to about 20 g / L. In some embodiments, the peptone comprises casein peptone and / or soy peptone. In some embodiments, the lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate-buffered saline, or any combination thereof. In some embodiments, the lysis buffer further comprises at least one additional nonionic surfactant, and optionally the at least one additional nonionic surfactant comprises a saponin.

[0027] In some embodiments, the lysis buffer does not contain additional nonionic surfactants. In some embodiments, the lysis buffer does not contain buffering agents. In some embodiments, the lysis buffer is acidic. In some embodiments, the lysis buffer does not contain saponins. In some embodiments, the lysis buffer contains Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA It does not contain one or more surfactants selected from the group consisting of 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-surfactant sulfobetaine (NDSB 201), amphipol (PMAL-C8), and methyl-β-cyclodextrin. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amide sulfobetaine-14, and C7BzO.In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol. In some embodiments, the lysis buffer has structure C. 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 The product does not contain polyoxyethylene surfactants (where ≠ 9-10 oxyethylene hydrophilic head groups). In some embodiments, at least one microorganism remains intact in the presence of SDA. In some embodiments, SDA does not damage at least one microorganism. [Brief explanation of the drawing]

[0028] [Figure 1] This document describes example data related to MALDI scores for Staphylococcus epidermidis isolated from positive blood cultures using different lysis agents. The MALDI scores for Staphylococcus epidermidis isolated from positive blood cultures using different lysis agents are shown. SAP represents saponin, and SDA represents the somatic digestive agent nonoxynol-9. A concentration of 0.52% (w / w) was used for each lysis agent. The score for identification approval is 1.8 for the Sepsityper database and 2.0 for the standard database. [Modes for carrying out the invention]

[0029] Detailed explanation The following detailed description refers to the accompanying drawings, which form part of the description. In the drawings, similar symbols typically identify similar components unless the context should interpret them otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to limit the scope. Other embodiments may be used, and other modifications may be made without departing from the spirit and scope of the subject matter presented herein. It will be readily apparent that the aspects of this disclosure can be arranged, replaced, combined, separated, and designed in a variety of different configurations, as generally described herein and as shown in the drawings, all of which are expressly intended herein and constitute part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated herein by reference with respect to the relevant technologies.

[0030] This specification discloses a method for processing a sample. In some embodiments, the method involves contacting a sample containing blood cells and at least one microorganism with a lysis buffer to produce a processed sample, wherein the lysis buffer contains a somatic digestive agent (SDA) capable of lysing the blood cells in the sample, and the SDA is given by the following formula 1

[0031] [ka]

[0032] The method comprises the step of lysing blood cells in a sample with a compound (wherein x is an integer from 2 to 20 and y is an integer from 6 to 11). In some embodiments, y is an integer from 8 to 10. In some embodiments, y is 8. In some embodiments, x is an integer from 5 to 15. In some embodiments, x is an integer from 8 to 12. In some embodiments, x is 9 or 10. In some embodiments, x is 9. In some embodiments, SDA is nonoxynol-9. This specification discloses compositions (e.g., kits). In some embodiments, the composition is a lysis buffer comprising a somatic cell digestive agent (SDA) capable of lysing blood cells, wherein the SDA is given by the following formula 1

[0033] [ka]

[0034] The lysis buffer comprises a compound of (wherein x is an integer from 2 to 20 and y is an integer from 6 to 11) and blood cells and / or their debris. In some embodiments, y is an integer from 8 to 10. In some embodiments, y is 8. In some embodiments, x is an integer from 5 to 15. In some embodiments, x is an integer from 8 to 12. In some embodiments, x is 9 or 10. In some embodiments, x is 9. In some embodiments, SDA is nonoxynol-9.

[0035] definition Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally interpreted by those skilled in the art to which this disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below. As used herein, the term “about” shall have its usual meaning when referring to measurable values ​​such as the amount, dose, time, temperature, etc., of the compounds or drugs disclosed herein (e.g., SDA), and shall also include differences of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified amount.

[0036] As used herein, the term “microorganism” is given its ordinary meaning and generally refers to, but is not limited to, single-celled organisms that can be grown and handled in a laboratory, including Gram-positive or Gram-negative bacteria, yeasts, fungi, parasites, and mollicutes. Non-specific examples of Gram-negative bacteria include the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, and Brevundimonas. The genera include Brevundimonas, Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella, Pasteurella, Providencia, and Legionella.Non-limiting examples of Gram-positive bacteria include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, and Propionibacterium. The genera include Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria, and Corynebacteria. Non-limiting examples of yeasts and fungi include the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces, and Trichosporon. Non-limiting examples of parasites include the following genera: Trypanosoma, Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, and Naegleria. Non-limiting examples of bacteria belonging to the class Moricutes include the genera Mycoplasma and Ureaplasma.

[0037] In some embodiments of the methods and compositions disclosed herein, microorganisms obtained from a sample or growth medium can be isolated and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term “isolate” is given its ordinary meaning and includes any sample of microorganisms in its original state or removed from the growth medium or culture medium, concentrated, or otherwise isolated. For example, in some embodiments, microorganisms may be isolated (e.g., as an isolation sample) from non-microorganisms or non-microorganismal components that might otherwise interfere with characterization and / or identification. An isolated microbial sample may include any collection or layer of microorganisms and / or their components, which may be more concentrated than the original sample or otherwise distinguished from the original sample, and can range from densely packed aggregates of microorganisms to diffuse layers of microorganisms. Microbial components that may be included in the isolation form or isolation sample include, but are not limited to, any combination of pili, flagella, trichomes, and capsules. Non-microbial components isolated from microorganisms include non-microbial cells (e.g., blood cells and / or other tissue cells) and / or any components thereof.

[0038] In some embodiments of the methods and compositions disclosed herein, microorganisms can be isolated from a sample or growth medium and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term “isolated” is given its ordinary meaning and includes any sample of microorganisms, whether in its initial state or at least partially purified from the growth medium or culture medium, and includes any non-microorganisms or non-microorganismal components contained therein. For example, in some embodiments, microorganisms are isolated (e.g., as an isolation sample) from non-microorganisms or non-microorganismal components that would otherwise interfere with characterization and / or identification. Non-microorganismal components isolated from microorganisms include non-microorganismal cells (e.g., blood cells and / or other tissue cells) and / or any components thereof.

[0039] In some embodiments of the methods and compositions disclosed herein, microorganisms obtained from a sample or growth medium can be pelletized and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term “pellet” is given its ordinary meaning and encompasses any sample of microorganisms that has been compressed or deposited into a mass of microorganisms. For example, microorganisms obtained from a sample can be compressed or deposited into a mass at the bottom of a tube by centrifugation or by other artisanally known methods. The term encompasses the collection of microorganisms (and / or their components) on the bottom and / or sides of the container after centrifugation. Microbial components that may be included in a pellet include, but are not limited to, any combination of pili, flagella, cilia, and capsules. In some embodiments, microorganisms can be pelletized (e.g., as a substantially purified microbial pellet) separated from non-microorganisms or non-microbial components that might otherwise hinder characterization and / or identification. Non-microbial components isolated from microorganisms include non-microbial cells (e.g., blood cells and / or other tissue cells) and / or any components thereof.

[0040] Dissolving buffer and method of use The various embodiments disclosed herein provide reagents and methods for the rapid isolation of intact and / or viable microbial cells from a sample containing Staphylococcus epidermidis (e.g., PBC). The resulting microbial pellets obtained using the various disclosed reagents and methods can be well free from interfering substances and can be used in identification methods, e.g., MALDI-TOF / MS, growth-based identification, and AST methods. This can enable rapid results without the need to subculture the microorganisms. The concentrated aggregates of viable microbial cells obtained by the various embodiments can be used for rapid ID systems, e.g., MALDI-TOF / MS, and direct inoculation of ID / AST tests using conventional or automated systems, e.g., the BD® Phoenix® ID / AST system. The various embodiments may also be applicable to other systems, molecular testing methods, e.g., polymerase chain reaction (PCR), and methods known to those skilled in the art.

[0041] Various embodiments disclosed herein provide reagents and methods for the rapid isolation of microbial cells, including Staphylococcus epidermidis, from positive blood culture samples. The resulting microbial pellets can be used in identification and / or growth-based methods, such as antimicrobial susceptibility testing. In some embodiments, the disclosed methods provide a process for the rapid isolation and concentration of viable microorganisms from PBC samples using only one sample preparation tube and centrifugation, while removing cellular debris from mammalian blood cells that may interfere with identification methods. Positive blood culture (PBC) samples can be obtained by methods known to those skilled in the art and not detailed herein. PBC samples may include, for example, samples determined to be positive for at least one microorganism by detection using the BD BACTEC® Instrumented Blood Culture System (Becton, Dickinson and Company). In one embodiment, the microorganism may include Gram-positive bacteria, Gram-negative bacteria, or yeast. In some embodiments, the microorganism is Staphylococcus epidermidis. The starting volume of the PBC sample is not limited to any particular maximum or minimum volume.

[0042] This specification provides methods and compositions comprising a novel and efficient hemocytolytic surfactant, SDA (Somatic Cell Digestive Agent). In some embodiments, SDA is an efficient hemocytolytic agent for isolating bacteria from positive blood cultures for rapid MALDI identification. In some embodiments, SDA is the nonionic surfactant nonoxynol-9. In some embodiments of the compositions and methods disclosed herein, SDA is used in a lysis buffer to lyse hemocytolytic cells to facilitate the isolation of bacterial cells from positive blood cultures for rapid identification by MALDI. In some embodiments, SDA can specifically disrupt hemocytolytic membranes without damaging bacterial cells. Currently available methods utilize saponins to lyse blood cells and isolate bacteria from positive blood cultures. However, MALDI identification for some bacterial strains yields very low scores, particularly for Staphylococcus epidermidis, which is often not identified. This identification failure is likely due to incomplete lysis of blood cells and / or high blood debris content. Using SDA instead of saponin in the lysis buffer to remove blood cells resulted in high MALDI scores with accurate identification (Figure 1). In some embodiments, SDA is very soluble in water. The advantage of using SDA as a lysis agent to isolate bacteria from positive blood cultures is that MALDI scores for difficult species, such as Staphylococcus epidermidis, may be higher than the cutoff value, leading to accurate species identification. Furthermore, the processing time using SDA compared to saponin may be nearly identical.

[0043] In some embodiments, a method for processing a sample is provided. In some embodiments, the method comprises the steps of producing a processed sample by contacting a sample containing blood cells and at least one microorganism with a lysis buffer, wherein the lysis buffer contains a somatic cell digestive agent (SDA) capable of lysing the blood cells in the sample, and thereby lysing the blood cells in the sample. In some embodiments, a composition (e.g., a kit) is provided. In some embodiments, the composition comprises a lysis buffer containing a somatic cell digestive agent (SDA) capable of lysing blood cells, and blood cells and / or their debris. In some embodiments, the SDA is a compound of the following formula 1.

[0044] [ka]

[0045] In some embodiments, x is an integer between 2 and 20. In some embodiments, x is an integer between 5 and 15. In some embodiments, x is an integer between 8 and 12. In some embodiments, x is 9 or 10. In some embodiments, x is 9. In some embodiments, y is an integer between 6 and 11. In some embodiments, y is an integer between 8 and 10. In some embodiments, y is 8. In some embodiments, SDA is nonoxynol-9. The concentrations of SDA in the lysis buffer, or in the final reaction volume when combined with the sample, are 0.001 g / L, 0.005 g / L, 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, and 40 g / L. , 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, or any number or range between these values, or approximately 0.001g / L, 0.005g / L, 0.01g / L, 0.05g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, or any number or range between these values, or at least 0.001g / L, 0.005g / L, 0.01g / L, 0.05g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, or any number or range between these values, or at most 0.001g / L, 0.005g / L, 0.01g / L, 0.It may be 0.5g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, or any number or range between these values. In the lysis buffer, or when combined with the sample, the concentration of SDA in the final reaction volume may be 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range (w / w) between these values, or approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range (w / w) between these values. It can be a range (w / w), or at least 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range (w / w) between any of these values, or at most 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range (w / w) between any of these values. The concentration of SDA in the lysis buffer, or in the final reaction volume when combined with the sample, is approximately 0.It may be 52% (w / w). In other embodiments, the percentages of the lysis buffer components disclosed herein are provided as %w / w, %m / v, %v / v, %m / w, %w / v, or variants thereof. The final concentration of SDA when combined with a sample is not limited as long as SDA is used at a concentration that causes hemolysis (or otherwise degradation) of at least some of the blood cells while leaving at least some of the microorganisms in the sample intact and / or viable. The final concentration of SDA upon contact with the sample may be 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range (in volume) between these values, or approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range (in volume) between these values. ) could be, or at least 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values, or at most 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range (in volume) between any of these values.

[0046] In some embodiments, the concentrations of the various components of the lysis buffer described herein represent the final concentrations of each component in the lysis buffer. In some embodiments, a sample (e.g., PBC) in a 1:1 volume ratio is mixed with the lysis buffer during the contact step, but other volume ratios are intended. Therefore, the concentrations of each component in the lysis buffer can be adjusted to account for changes in the volume ratio of the lysis buffer to the PBC sample in order to achieve the desired final concentrations of the components of the lysis buffer when mixed with the sample (e.g., PBC). The method for isolating microorganisms from a sample suspected to contain at least one microorganism, such as a PBC sample, as described herein, can utilize various lysis buffers containing SDA designed to rapidly produce a pellet of viable microorganisms that can be used for various downstream testing methods, such as identification by MALDI-TOF / MS, growth-based phenotypic assays, and AST testing. In some embodiments, the method includes the step of adding a portion of the sample to a lysis buffer containing SDA to form a mixture. In some embodiments, the volume ratio of the sample to the SDA-containing lysis buffer is approximately 1:1. The mixture can be incubated for a time to lyse the blood cells in the PBC sample.

[0047] In some embodiments, the volume ratio of the sample to the SDA-containing lysis buffer is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24. , 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64 , 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400 , 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or any number or range between any two of these values, or approximately 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:3 5, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1: 67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, The ratios may be 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or any number or range between any two of these values. In some embodiments, the volume ratio of the sample to the SDA-containing lysis buffer may be 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38: 1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1 ,74:1,75:1,76:1,77:1,78:1,79:1,80:1,81:1,82:1,83:1,84:1,85:1,86:1,87:1,88:1,89:1,90:1,91:1,92:1,93:1,94:1,95:1,96:1,97:1,98:1,99:1,100:1,200:1,300:1,400:1,500:1,600:1,700:1,800 :1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of these values, or approximately 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35 :1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 6 7:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, It could be 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of these values.

[0048] In some embodiments, the sample is in contact with the lysis buffer two or more times. For example, the sample may be in contact with the lysis buffer two, three, four, five, six, seven, eight, nine, or ten times during the sample preparation process. In some embodiments, the sample may be in contact with the lysis buffer, centrifuged to produce a pellet, which may then be resuspended, and the resuspended pellet may undergo one or more further contact steps with the lysis buffer. In some embodiments, the contact steps include an incubation time lasting 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, 200, 300, 400, 500, or a number or range (minutes) between any of these values.

[0049] Some embodiments of the methods and compositions provided herein are useful for the isolation, characterization, and / or identification of microorganisms from complex samples such as blood-containing cultures. In some embodiments, the methods disclosed herein enable faster characterization and / or identification of microorganisms than currently available methods, resulting in faster diagnosis (e.g., of a subject with or suspected of having sepsis) and characterization / identification of contaminants (e.g., food and pharmaceuticals). The steps included in the disclosed methods are performed within a very short timeframe, from obtaining the sample to characterizing / identifying the microorganisms, to obtain clinically relevant actionable information. In certain embodiments, the disclosed methods may be performed in less than about 120 minutes, e.g., less than about 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 minute, or within a range or number (minutes) of any of these values. In some embodiments, the speed of the methods disclosed herein represents an improvement over currently available methods. The disclosed methods can be used to characterize and / or identify any of the microorganisms described herein. In some embodiments, the disclosed methods can be fully automated, thereby reducing the risk of handling infectious material and / or contaminating samples.

[0050] Samples that can be tested by the methods disclosed herein (e.g., test samples) include not only clinical and non-clinical samples in which the presence and / or growth of microorganisms is suspected or may be suspected, but also samples of materials that are routinely or occasionally tested for the presence of microorganisms. The amount of sample used can vary considerably due to the versatility and / or sensitivity of the methods disclosed herein. Sample preparation can be carried out by any number of techniques known to those skilled in the art, but one advantage of the methods disclosed is that complex sample types, such as blood, body fluids, and / or other opaque substances, can be tested by directly using the system with little or no extensive pretreatment. In some embodiments, the sample is taken from a culture. In some embodiments, the sample is taken from a microbial culture (e.g., a blood culture). In some embodiments, the sample is suspected of containing microorganisms or is known to contain microorganisms. Clinical samples that can be tested include, but are not limited to, any type of sample typically tested in a clinical or research laboratory, such as blood, serum, plasma, blood fractions, synovial fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissue homogenates, bone marrow aspirate, bone homogenates, sputum, aspirate, swabs and swab rinses, and other body fluids. In some embodiments, clinical samples are cultured, and cultured samples are used.

[0051] The compositions and methods disclosed herein find applications not only in research but also in veterinary and medical applications. Suitable subjects from which clinical samples can be obtained are generally mammalian subjects, but may be any animal. The term “mammal” as used herein is given in its ordinary sense, but does not include, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, and rodents (e.g., rats or mice). Human subjects include neonates, infants, young, adult, and elderly subjects. Subjects from which samples can be obtained include, but do not include, mammals, birds, reptiles, amphibians, and fish. Nonclinical samples that can be tested include, but are not limited to, food, beverages, pharmaceuticals, cosmetics, water (e.g., drinking water, non-drinking water, and wastewater), seawater ballast, air, soil, sewage, plant matter (e.g., seeds, leaves, stems, roots, flowers, fruits), blood products (e.g., platelets, serum, plasma, leukocyte fractions), donor organ or tissue samples, biological weapons samples, and other materials. The methods disclosed herein can be used for real-time testing to monitor contamination levels, process control, quality control, etc., in industrial environments. In some embodiments, nonclinical samples are cultured, and cultured samples are used.

[0052] In some embodiments, the sample is obtained from a subject (e.g., a patient) who is infected with or suspected of being infected with bacteria. In some embodiments, the subject is suffering from or suspected of having sepsis, e.g., bacteremia or fungemia. The sample may be a blood sample obtained directly from the subject. The sample may be from a blood culture grown from a sample of the patient's blood. The blood culture sample may be from a positive blood culture, e.g., a blood culture showing the presence of microorganisms. In some embodiments, the sample is taken from a positive blood culture within a short time after it became positive, e.g., within about 6 hours, e.g., within about 5, 4, 3, or 2 hours, or within about 60 minutes, e.g., within about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 minute, or within a range or number between those values. In some embodiments, the sample is taken from a culture in which the microorganisms are in the logarithmic growth phase. In some embodiments, the sample is taken from a culture in which the microorganisms are in the quiescent phase.

[0053] Various embodiments of the disclosed method can provide high sensitivity for the detection, characterization, and / or identification of microorganisms. Various embodiments of the disclosed method can enable detection, characterization, and / or identification without the need to first go through the step of isolating the microorganism by growing it on a solid or semi-solid medium and sampling the growing colonies. Thus, in some embodiments, the sample is not from colonies of microorganisms (e.g., bacteria, yeast, or mold) grown on a solid or semi-solid surface. In some embodiments, the sample volume is large enough to produce an isolated sample of microorganisms or a pellet of microorganisms that can be examined after the separation / isolation step of the method disclosed herein has been performed. The appropriate volume depends on the origin of the sample and the expected level of microorganisms in the sample. For example, a positive blood culture may require a smaller volume of blood culture than a drinking water sample because it contains a higher level of microorganisms per unit volume than a drinking water sample to be tested for contamination. Generally, the sample size may be less than about 50 ml, e.g., about 40, 30, 20, 15, 10, 5, 4, 3, or 2 ml, or a range or number between those values. In some embodiments, the sample size may be about 1 ml, e.g., about 0.75, 0.5, or 0.25 ml, or a range or number between those values. In some embodiments where separation is performed on a microscale, the sample size may be less than approximately 200 μl (e.g., less than approximately 150, 100, 50, 25, 20, 15, 10, or 5 μl, or a range or number (μl) between those values). In some embodiments (e.g., when the sample is expected to contain a small number of microorganisms), the sample size may be 100 ml or more, e.g., 250, 500, 750, or 1000 ml or more, or a range or number (ml) between those values.

[0054] The sample may be derived from a blood culture of a subject who is infected or suspected of being infected. The sample may include a positive blood culture sample determined to contain at least one microorganism. The at least one microorganism may be selected from the group including Gram-positive bacteria, Gram-negative bacteria, and yeast. The at least one microorganism may be Staphylococcus epidermidis. The at least one microorganism may be Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and / or Streptococcus pneumoniae. The contact step may include sonication, osmotic shock, chemical treatment, or any combination thereof. The lysis buffer may contain one or more proteinases and / or one or more nucleases. The method may include a step of isolating at least one microorganism from the treated sample to produce at least one isolated microorganism. The step of isolating at least one microorganism from the treated sample may include a step of separating at least one microorganism from lysed blood cells. The step of isolating at least one microorganism from lysed blood cells may include the steps of centrifuging the treated sample to produce a pellet and a supernatant, and discarding the supernatant while retaining the pellet containing at least one isolated microorganism. The method may include the steps of preparing a plate pure culture from at least one isolated microorganism and analyzing the microorganism obtained from this plate pure culture. The method may include the steps of preparing an inoculum from at least one isolated microorganism and analyzing at least one microorganism obtained from this inoculum. Methods, apparatus, compositions, and systems for the isolation and identification of microorganisms from a sample (e.g., a positive blood culture) are described in U.S. Patents 10,059,975 and 9,180,448, which are incorporated herein by reference in their entirety.

[0055] Some embodiments described herein can be used in conjunction with the composition and method for rapid processing and identification of microorganisms from positive blood cultures described in U.S. Patent No. 9,631,221, which is incorporated herein by reference in its entirety. The method may include the steps of: depositing at least a portion of a pellet containing at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of at least one microorganism by mass spectrometry; optionally drying the deposited sample; treating the deposited sample with a volatile acid solution, wherein the volume percentage of the volatile acid may be at least 70% of the volatile acid solution combined with the deposited sample; optionally drying the treated deposited sample; placing a matrix on top of the treated deposited sample; and optionally drying the treated deposited sample. The volatile acid solution may be an aqueous solution of a volatile acid or a volatile solution in an organic solvent. In some embodiments, the volatile acid solution may be an aqueous formic acid solution in volume percent of about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or a range or number of values ​​between these values ​​when combined with the deposited sample. The method may include the step of treating the deposited sample with an organic solvent and drying the deposited sample before treating the deposited sample with the volatile acid solution. The organic solvent may include ethanol, methanol, isopropanol, acetonitrile, acetone, ethyl acetate, or any combination thereof.

[0056] Some embodiments described herein can be used in conjunction with various reagents (e.g., choline-containing solutions) and methods for rapidly isolating viable microbial cells from positive blood culture samples for use in downstream analyses such as identification and antimicrobial susceptibility testing, as described in U.S. Patent No. 8,603,769, which is incorporated herein by reference in its entirety. While the applicant does not wish to be bound by any particular theory, the addition of a choline-containing solution in the presence of a solubility component (e.g., SDA) of the buffer disclosed herein may inhibit, prevent, and / or mitigate microbial autodegradation. The method may include the step of contacting the sample with a choline-containing solution before, during, and / or after contact with the solubility buffer. In some embodiments, the compositions disclosed herein further include a choline-containing solution. The choline-containing solution is expressed by the following formula 2

[0057] [ka]

[0058] (In the formula, R 1 , R 2 , and R 3 (where X independently represents a group selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and X represents the loaded electrolytic group.) It may contain at least one quaternary ammonium salt containing an N,N,N-trimethylethanolammonium cation, selected from the group consisting of the following. In some embodiments, X is selected from the group consisting of chlorides, fluorides, nitric acid, and bicarbonate. The choline-containing solution may contain choline chloride. The choline-containing solution may contain phosphorylcholine. When in contact with the sample, the final concentration of choline by volume may be 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range between these values, or approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or any number or range between these values. It may be a range, or at least 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values, or at most 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values. In other embodiments, the percentages of the lysis buffer components disclosed herein are provided as %w / w, %m / v, %v / v, %m / w, %w / v, or variations thereof.The concentration of choline in the sample during the contact step may be, by volume, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values, or approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values. It may be a range, or at least 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values, or at most 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, or a number or range between any of these values. The concentration of choline in the sample during the contact step may be in the range of approximately 0.25% by volume to approximately 10% by volume (e.g., 0.25%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range or number between any two of these values). The contact step may include incubating the sample with the choline-containing solution for up to 20 minutes. The incubation temperature may be room temperature.

[0059] The lysis buffers disclosed herein may contain one or more components that help stabilize microorganisms, allow the lysis reagent to lyse blood cells, and / or remove interfering cell debris. Some embodiments described herein can be used with various reagents and methods for rapidly isolating viable microbial cells from positive blood culture samples, as described in U.S. Patent No. 10,519,482, which is incorporated herein by reference in its entirety. The lysis buffer may contain an antifoaming agent. In some embodiments, the lysis buffer does not contain an antifoaming agent. The lysis buffer may contain at least one thiol. At least one thiol may include L-cysteine ​​HCl, sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid, mercaptoethanol, mercaptoethanesulfonic acid, thioglycerol, or any combination thereof.The concentration of at least one thiol in the lysis buffer is 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number between these values. The values ​​may be a range (g / L), or approximately 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. It may be L) or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. It may be, or at most, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. At least one thiol may contain L-cysteine ​​and / or sodium thioglycolate.The concentration of L-cysteine ​​and / or sodium thioglycolate in the lysis buffer may be 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or any number or range (g / L) between these values, or approximately 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or any number or range (g / L) between these values. The soluble buffer may be a range (g / L), or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or any number or range (g / L) between these values, or at most 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or any number or range (g / L) between these values. The solubilizing buffer may contain ammonium chloride.The concentration of ammonium chloride in the lysis buffer is 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range between these values. It may be in the range (g / L), or approximately 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. It may be, or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. It may be, or at most 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range (g / L) between any of these values.

[0060] The lysis buffer may contain a nutrient base solution comprising one or more of the following: casein peptone at a concentration of approximately 8 g / L to approximately 35 g / L, sodium chloride at a concentration of approximately 2 g / L to approximately 10 g / L, soy peptone at a concentration of approximately 1.5 g / L to approximately 15 g / L, and potassium phosphate at a concentration of approximately 0.5 g / L to approximately 5 g / L, along with at least one other nutrient. The at least one other nutrient may include a nutrient medium. The concentration of the nutrient medium in the lysis buffer may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range (g / L) between these values, or approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range (g / L) between these values. The nutrient may be, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values, or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. At least one other nutrient may be included in the nutrient medium, comprising one or more of i) tryptone, ii) soy, iii) NaCl, iv) dipotassium phosphate (K2HPO4), and v) glucose. The lysis buffer may comprise one or more of the nutrient medium, isotonic buffer, peptone, and salt. The nutrient medium may comprise Trypticase Soy Medium. Isotonic buffers may include sodium phosphate, potassium phosphate, phosphate-buffered saline, physiological saline, or any combination thereof.The concentration of isotonic buffer in the lysis buffer may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range (g / L) between these values, or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or any number or range (g / L) between these values. It may be, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values, or at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or a number or range (g / L) between any of these values. Peptone may include casein peptone and / or soy peptone. The lysis buffer may contain sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate-buffered saline, or any combination thereof. In some embodiments, the lysis buffer may contain at least one additional nonionic surfactant (e.g., saponin). In some embodiments, the lysis buffer does not contain an additional nonionic surfactant.

[0061] The method may include a step of identifying at least one microorganism. The step of identifying at least one microorganism may include mass spectrometry, phenotypic identification, antimicrobial susceptibility testing, molecular testing, or any combination thereof. Mass spectrometry may include electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, or ESI-MS / (MS). nMatrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), silicon desorption / ionization (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCJ-MS / MS, APCI-(MS) n , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS) n This can include one or more of the following: quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero. Mass spectrometry may include MALDI-TOF-MS.

[0062] In some embodiments, SDA does not damage at least one microorganism. At least one microorganism may remain intact in the presence of SDA. In some embodiments, the method yields a MALDI score at least 1% higher (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, or any number or range between these values) compared to a comparative method using a lysis buffer without SDA. In some embodiments, the comparison method utilizes a lysis buffer containing saponins. In some embodiments, the lysis buffer selectively dissolves at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or a number or range (%) between any of these values. In some embodiments, the ratio of lysed blood cells to lysed cells of at least one microbial species after the contact step is 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1 ,39:1,40:1,41:1,42:1,43:1,44:1,45:1,46:1,47:1,48:1,49:1,50:1,51:1,52:1,53:1,54:1,55:1,56:1,57:1,58:1,59:1,60:1,61:1,62:1,63:1,64:1,65:1,66:1,67:1,68:1,69:1,70:1,71:1,72:1,73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800: It could be 1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of these values, or approximately 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35 :1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 6 7:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of these values. In some embodiments, the ratio of lysed blood cells to lysed cells of at least one microbial species after the contact step may be at least 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37: 1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1 ,72:1,73:1,74:1,75:1,76:1,77:1,78:1,79:1,80:1,81:1,82:1,83:1,84:1,85:1,86:1,87:1,88:1,89:1,90:1,91:1,92:1,93:1,94:1,95:1,96:1,97:1,98:1,99:1,100:1,200:1,300:1,400:1,500:1, It can be 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1, or at most 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 3 4:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95: The ratios may be 1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the cells of at least one microbial species remain intact and / or viable after the contact step.

[0063] The viable and / or intact microbial pellets resulting from the various embodiments described herein can be used to prepare common samples for various downstream testing methods, including identification by mass spectrometry, e.g., MALDI-TOF / MS identification, identification based on phenotypic growth, e.g., Phoenix® ID, and AST testing, e.g., Phoenix® AST testing. Furthermore, the entire method can be performed in a single sample tube without requiring sample transfer between multiple tubes. Thus, the methods described herein can be readily adapted to automated systems. The yield can be improved by increasing the number of microorganisms in the starting volume and employing techniques such as larger PBC sample volumes, multiple aliquots of PBC samples, and multiple spins. Furthermore, these methods provide a rapid sample preparation method and are easily automated. In addition, the methods and buffers described herein expose blood cells to lysis, remove interfering substances from the PBC sample, and result in a high yield of viable microorganisms. In one embodiment, the yield of viable microbial pellets can be increased by increasing the starting volume of the PBC sample and / or by performing an isolation method on several aliquots from one PBC sample and combining the resulting microbial pellets into a single sample.

[0064] In one embodiment, isolated microorganisms are treated in preparation for downstream testing. This includes, for example, resuspending at least a portion of the isolated microorganisms in a fluid, such as water, OG, BD Phoenix® ID medium, or a nonionic surfactant. In one embodiment, isolated microorganisms are prepared for identification by mass spectrometry by resuspending the isolated microorganism pellet in solution and depositing a portion of the resuspended pellet onto a MALDI-TOF MS plate, or by directly depositing a portion of the isolated microorganisms onto a MALDI-TOF MS plate without first resuspending the pellet in solution. In another embodiment, isolated microorganism pellets are prepared for BD Phoenix® ID / AST testing by resuspending the isolated microorganism pellet in solution and adjusting this suspension to a specific concentration of about 0.5 McFarland Standard. Further methods for preparing isolated microorganisms for downstream analysis are known to those skilled in the art and are not described herein in detail.

[0065] Isolated microorganisms can be used for several downstream analyses, including microbial identification (e.g., mass spectrometry, phenotypic or molecular identification methods) and AST testing. Many technically known manual and automated AST systems are applicable, including BD Phoenix® ID / AST, disk diffusion (Sensi-Disc), agar dilution, and micro / macro tube dilution. Identification methods and AST testing are well known to those skilled in the art and are not detailed herein. Further downstream tests include, for example, enzymatic reactions, biochemical reactions, various molecular or phenotypic identification systems, and / or various phenotypic identification systems or methods utilizing growth-based identification schemes. These may be used to detect resistance markers that provide protection for bacterial isolates from specific antimicrobial agents and classes. The various methods described herein further include the preparation of plate cultures or single inoculum from isolated microorganisms. Methods for preparing plate cultures or inoculum are known to those skilled in the art. If further downstream testing is required, plate cultures or inoculum can be prepared to obtain a sufficient quantity of sample. A portion of the isolated (e.g., pelletized) microorganisms obtained by the disclosed method can be inoculated into BD Phoenix® ID medium (Becton, Dickinson and Company). A portion of the inoculation material can be inoculated into the AST portion of the BD Phoenix® ID / AST panel (Becton, Dickinson and Company). The BD Phoenix® ID / AST system is described in, for example, U.S. Patents 5,922,593, 6,096,272, 6,372,485, 6,849,422, and 7,115,384, the entire contents of which are incorporated herein by reference.

[0066] In some embodiments, the lysis buffer does not contain a buffer. In some embodiments, the lysis buffer is acidic. In some embodiments, the identification of at least one microorganism does not involve spectroscopic measurements (e.g., intrinsic fluorescence spectroscopy). In some embodiments, the method does not involve density gradient centrifugation. In some embodiments, the lysis buffer does not contain saponins. In some embodiments, the lysis buffer is Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA It does not contain one or more surfactants selected from the group consisting of 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-surfactant sulfobetaine (NDSB 201), amphipol (PMAL-C8), and methyl-β-cyclodextrin. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers.In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO. In some embodiments, the lysis buffer does not contain one or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol. In some embodiments, the lysis buffer has structure C. 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 It does not contain polyoxyethylene surfactants (which represent 9 to 10 oxyethylene hydrophilic head groups). [Examples]

[0067] Examples Some aspects of the above embodiments are further disclosed in the following embodiments, which are not intended to limit the scope of this disclosure. Example 1 MALDI score for Staphylococcus epidermidis isolated from positive blood cultures using different lysis agents. This example demonstrates the identification of Staphylococcus epidermidis isolated from positive blood cultures using the sample processing methods and compositions provided herein. Figure 1 shows example data regarding MALDI scores for Staphylococcus epidermidis isolated from positive blood cultures using different lysis agents, namely saponins (SAP), nonoxynol-9 (somatic digestive agent (SDA)), and combinations thereof. Each lysis agent was used at a concentration of 0.52% (w / w). A score of 1.8 or higher is required for approved identification in the Sepsityper database, and 2.0 or higher in the standard database. Surprisingly, using SDA as the lysis buffer resulted in higher MALDI scores and more accurate identification. Using saponins, currently used lysis agents, resulted in lower MALDI scores. SDA is commonly used as a spermicide by interacting with lipids in the acrosome membrane and spermatospinal flap. Although SDA is commonly used as a spermicide, it was surprisingly found to be able to dissolve blood cells very efficiently.

[0068] In at least some of the embodiments described above, one or more elements used in one embodiment may be interchangeably used in another embodiment, but only if such interchangeability is technically feasible. Those skilled in the art will see that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes shall fall within the scope of the subject matter as defined by the appended claims. In substantially any use of plural and / or singular terms herein, a person skilled in the art may substitute plural for singular and / or singular for plural as appropriate to the context and / or application. Various singular / plural permutations are explicitly shown herein for clarity. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise specified.

[0069] In general, those skilled in the art will understand that the terms used herein, and in particular in the appended claims (e.g., the text of the appended claims), are generally "open" terms (for example, the term "including" should be interpreted as "including but not limited to," the term "having" as "having at least," and the term "includes" as "includes but is not limited to," etc.). Furthermore, those skilled in the art will understand that where a specific number of introductory claims are intended, this intention is explicitly stated in the claims, and where there is no such intention, there is no such intention in the absence of such enumeration. For example, to aid understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more)" to introduce a list of claims. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim enumeration by the indefinite article "a" or "an" limits any particular claim containing the introduced claim enumeration to embodiments containing only one such enumeration, even when the claim contains the prefix phrase "one or more" or "at least one" and the indefinite article "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same is true for the use of the definite article used to introduce a claim enumeration. Furthermore, even if a specific number of introduced claim enumerations are explicitly stated, a person skilled in the art will recognize that such enumerations should be interpreted as meaning at least the number of enumerations (for example, a bare enumeration of "two enumerations" without other modifiers means at least two enumerations, or two or more enumerations). Furthermore, when a similar convention is used to mean "at least one of A, B, and C, etc.," the configuration is generally intended to be interpreted as such by those skilled in the art (for example, "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together).Where a similar convention is used to "at least one of A, B, or C, etc.," the configuration is generally intended to be as a convention would be interpreted by those skilled in the art (for example, "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together). Furthermore, those skilled in the art will understand that, in practice, any disjunctive word and / or phrase representing two or more alternative terms in the specification, claims, or drawings should be understood as intending the possibility of including one of those terms, either of those terms, or both.

[0070] Furthermore, if the characteristics or aspects of the disclosure are described in relation to the Markush group, a person skilled in the art would recognize that, as a result, the disclosure is also described in relation to any individual member or subgroup of members of the Markush group. As those skilled in the art will understand, for all purposes, including the provision of written detail, all scopes disclosed herein encompass all possible subscopes and combinations thereof. Each described scope is readily recognizable as fully representing and enabling decomposition into at least equivalent halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope described herein can be readily decomposed into a lower third, middle third, upper third, etc. Furthermore, as those skilled in the art will understand, all language such as “up to,” “at least,” “greater than,” and “less than” includes the listed number and refers to a scope that can subsequently be decomposed into the aforementioned subscopes. Finally, as those skilled in the art will understand, each scope encompasses its individual members. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be obvious to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit the scope, and the true scope and spirit are shown by the following claims. Another aspect of the present invention may be as follows: [1] A step of producing a processed sample by contacting a sample containing blood cells and at least one microorganism with a lysis buffer, wherein the lysis buffer contains a somatic cell digestive agent (SDA) capable of lysing the blood cells in the sample, and the SDA is given by the following formula 1 [ka] (In the formula, x is an integer between 2 and 20, and y is an integer between 6 and 11) This is a compound of the form, This step dissolves the blood cells in the sample. A method for processing a sample, including the processing of the sample. [2] The method according to [1], wherein y is an integer between 8 and 10, and in some cases y is 8. [3] The method according to any one of the above [1] to [3], wherein x is an integer between 5 and 15, and in some cases x is an integer between 8 and 12, and in other cases x is 9 or 10. [4] The method according to [1], wherein the SDA is nonoxynol-9. [5] The method according to any one of the above [1] to [4], wherein the concentration of SDA in the lysis buffer is about 0.01 g / L to about 10 g / L. [6] The method according to any one of the above [1] to [5], wherein the concentration of SDA in the lysis buffer is approximately 0.01% (w / w) to approximately 10% (w / w), possibly approximately 0.01% (w / w) to approximately 1% (w / w), and possibly approximately 0.52% (w / w). [7] The method according to any one of the above [1] to [3], wherein the sample is derived from a blood culture of a subject suspected of being infected. [8] The method according to any one of the above [1] to [7], wherein the sample includes a positive blood culture sample in which it has been determined that the sample contains at least one microorganism. [9] The method according to any one of the above [1] to [8], wherein the at least one microorganism is selected from the group including Gram-positive bacteria, Gram-negative bacteria and yeasts, and the at least one microorganism may include one or more of the following: Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pneumoniae.

[10] The method according to any one of the items [1] to [9], wherein the contact step includes ultrasonic treatment, osmotic shock, chemical treatment, or any combination thereof.

[11] The method according to any one of the above [1] to

[10] , wherein the lysis buffer comprises one or more proteinases and / or one or more nucleases.

[12] The process includes the step of isolating at least one microorganism from the processed sample to produce at least one isolated microorganism, and optionally the step of isolating at least one microorganism from the processed sample includes the step of separating at least one microorganism from lysed blood cells, and optionally the step of separating at least one microorganism from lysed blood cells, The steps include: centrifuging the processed sample to produce a pellet and a supernatant; The method according to any one of the claims [1] to

[11] , comprising the step of discarding the supernatant while retaining the pellet containing at least one isolated microorganism.

[13] The method according to

[12] , further comprising the step of preparing a plate pure culture from at least one isolated microorganism and analyzing the microorganism obtained from the plate pure culture.

[14] The method according to any one of the items [1] to

[13] , further comprising the steps of preparing an inoculum from the at least one isolated microorganism and analyzing the at least one microorganism obtained from the inoculum.

[15] A step of depositing at least a portion of the pellet containing at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of the at least one microorganism by mass spectrometry, Depending on the circumstances, the step of drying this deposited sample may be necessary. A step of treating the deposited sample with a volatile acid solution, wherein the volume percentage of the volatile acid solution is at least 70% of the volatile acid solution combined with the deposited sample. Depending on the circumstances, the process may involve drying the treated deposited sample, The steps include placing a matrix on the treated deposition sample, Depending on the circumstances, the process may involve drying the treated deposition sample. The method described in any one of the above [1] to

[14] , further including:

[16] The method according to

[15] , wherein the volatile acid solution is an aqueous solution of a volatile acid or a volatile solution in an organic solvent, and the organic solvent may include ethanol, methanol, isopropanol, acetonitrile, acetone, ethyl acetate, or any combination thereof.

[17] The method according to any one of the above

[15] to

[16] , wherein the volatile acid solution, when combined with the deposited sample, is an aqueous formic acid solution with a volume percentage of about 70% to about 90%.

[18] The method according to any one of the items

[15] to

[17] , further comprising the step of treating the deposited sample with an organic solvent and drying the deposited sample before treating the deposited sample with a volatile acid solution.

[19] The method according to any one of the items [1] to

[18] , further comprising the step of contacting the sample with a choline-containing solution before, simultaneously with, and / or after contact with the lysis buffer.

[20] The choline-containing solution is given by the following formula 2

change

[19] , comprising at least one quaternary ammonium salt containing an N,N,N-trimethylethanolammonium cation, selected from the group consisting of the above.

[21] The method according to

[20] , wherein X is selected from the group consisting of chlorides, fluorides, nitric acid, and bicarbonate.

[22] The method according to any one of the above

[19] to

[21] , wherein the choline-containing solution comprises choline chloride, phosphorylcholine, or both.

[23] The method according to any one of the above

[19] to

[22] , wherein the final concentration of choline when in contact with the sample is about 0.25% by volume or about 1% by volume or more.

[24] The method according to any one of the claims

[19] to

[23] , wherein the concentration of choline in the sample during the contact step is in the range of about 0.25% by volume to about 10% by volume, and optionally about 1.8% by volume to about 4% by volume.

[25] The method according to any one of

[19] to

[24] , wherein the contact step comprises incubating the sample with the choline-containing solution for up to 20 minutes, and the incubation temperature is room temperature.

[26] The method according to any one of the above [1] to

[25] , wherein the solubilating buffer further comprises an antifoaming agent.

[27] The method according to any one of the above [1] to

[25] , wherein the solubilating buffer does not contain an antifoaming agent.

[28] The method according to any one of the claims [1] to

[27] , wherein the lysis buffer further comprises at least one thiol, which may comprise L-cysteine ​​HCl, sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid, mercaptoethanol, mercaptoethanesulfonic acid, thioglycerol, or any combination thereof, and which may further comprise the concentration of the at least one thiol in the lysis buffer at about 0.005 g / L to 4 g / L.

[29] The method according to

[28] , wherein the at least one thiol comprises L-cysteine ​​in the lysis buffer at a concentration of about 0.01 g / L to about 2.5 g / L, and / or sodium thioglycolate in the lysis buffer at a concentration of about 0.01 g / L to about 2.5 g / L.

[30] The method according to any one of the above [1] to

[29] , wherein the solubilating buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilating buffer is about 0.01 g / L to about 80 g / L.

[31] The method according to any one of [1] to

[30] , wherein the lysis buffer further comprises a nutritional base solution containing one or more of the following in the lysis buffer: casein peptone at a concentration of about 8 g / L to about 35 g / L, sodium chloride at a concentration of about 2 g / L to about 10 g / L, soy peptone at a concentration of about 1.5 g / L to about 15 g / L, and potassium phosphate at a concentration of about 0.5 g / L to about 5 g / L, and at least one other nutrient.

[32] The method according to

[31] , wherein the nutrient medium contains at least one other nutrient in the lysis buffer at a concentration of about 10 g / L to about 50 g / L.

[33] The above at least one other nutrient is i) tryptone, ii) soy, iii) NaCl, iv) dipotassium phosphate (K 2 Hpo 4 The method according to any one of the above

[31] to

[32] , comprising a nutrient medium containing one or more types of glucose, and v) a glucose.

[34] The method according to any one of the above [1] to

[33] , wherein the lysis buffer further comprises one or more of a nutrient medium, an isotonic buffer, a peptone, and a salt, and the concentration of the nutrient medium in the lysis buffer is optionally about 10 g / L to about 50 g / L.

[35] The method according to any one of the above

[32] to

[34] , wherein the nutrient medium comprises Triptycase soy medium.

[36] The method according to any one of the above

[34] to

[35] , wherein the isotonic buffer comprises sodium phosphate, potassium phosphate, phosphate buffered saline, physiological saline, or any combination thereof, and the concentration of the isotonic buffer in the dissolution buffer is optionally about 1 g / L to about 20 g / L.

[37] The method according to any one of the items

[34] to

[36] , wherein the peptone comprises casein peptone and / or soy peptone.

[38] The method according to any one of the above [1] to

[37] , wherein the lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof.

[39] The method according to any one of the claims [1] to

[38] , wherein the solubilating buffer further comprises at least one additional nonionic surfactant, and optionally the at least one additional nonionic surfactant comprises a saponin.

[40] The method according to any one of the above [1] to

[38] , wherein the solubilating buffer does not contain an additional nonionic surfactant.

[41] The method according to any one of the items [1] to

[40] , further comprising the step of identifying at least one microorganism.

[42] The method according to

[41] , wherein the identification of at least one microorganism comprises mass spectrometry, phenotypic identification, antimicrobial susceptibility testing, molecular testing, or any combination thereof.

[43] Mass spectrometry includes electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, and ESI-MS / (MS). n Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), silicon desorption / ionization (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCJ-MS / MS, APCI-(MS) n , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS) n The method according to any one of the above

[15] to

[42] , comprising one or more of quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.

[44] The method according to any one of the items [1] to

[43] , wherein the SDA does not damage the at least one microorganism.

[45] The method according to any one of the claims [1] to

[44] , wherein the at least one microorganism remains intact in the presence of the SDA.

[46] The method according to any one of the items [1] to

[45] , wherein the method yields a MALDI score at least 5% higher than a comparative method using a lysis buffer that does not contain the SDA.

[47] The method according to

[46] , wherein the comparison method utilizes a solubilating buffer containing saponin.

[48] ​​The method according to any one of the claims [1] to

[47] , wherein the lysis buffer selectively dissolves at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the blood cells in the sample.

[49] The method according to any one of the claims [1] to

[48] , wherein the ratio of lysed blood cells to lysed cells of the at least one microorganism after the contact step is at least about 2:1.

[50] The method according to any one of the items [1] to

[49] , wherein after the contact step, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the cells of the at least one microorganism remain intact and / or viable.

[51] The method according to any one of the above [1] to

[50] , wherein the lysis buffer does not contain a buffering agent.

[52] The method according to any one of the above [1] to

[51] , wherein the lysis buffer is acidic.

[53] The method according to any one of the items

[41] to

[52] , wherein the identification of at least one microorganism does not include spectroscopic measurement, and the spectroscopic measurement is intrinsic fluorescence spectroscopy.

[54] The method according to any one of the above [1] to

[53] , wherein the method does not include density gradient centrifugation.

[55] The lysis buffer is (a) Saponins; (b) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-surfactant sulfobetaine (NDSB 201), amphipol (PMAL-C8), and one or more surfactants selected from the group consisting of methyl-β-cyclodextrin; (c) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether - one or more surfactants selected from this group; (d) Sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C 7 One or more surfactants selected from the group consisting of BzO; (e) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and / or (f) Structure C 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 Polyoxyethylene surfactant containing 9-10 oxyethylene hydrophilic head groups. The method described in any one of the above items [1] to

[54] , except that which does not include the method described in the above items.

[56] A lysis buffer containing a somatic cell digestive agent (SDA) capable of lysing blood cells, wherein the SDA is defined by the following formula 1

change

[57] The composition according to

[56] , wherein y is an integer from 8 to 10, and in some cases y is 8.

[58] The composition according to any one of the above items

[56] to

[57] , wherein x is an integer between 5 and 15, and in some cases x is an integer between 8 and 12, and in other cases x is 9 or 10.

[59] The composition according to

[56] , wherein the SDA is nonoxynol-9.

[60] The composition according to any one of the above items

[56] to

[59] , wherein the concentration of SDA in the lysis buffer is about 0.01 g / L to about 10 g / L.

[61] The composition according to any one of the above

[56] to

[60] , wherein the concentration of SDA in the lysis buffer is about 0.01% (w / w) to about 10% (w / w), optionally about 0.01% (w / w) to about 1% (w / w), and optionally about 0.52% (w / w).

[62] The composition according to any one of

[56] to

[61] , wherein the lysis buffer comprises one or more proteinases and / or one or more nucleases.

[63] Formula 2 below

change

[56] to

[62] .

[64] The composition according to

[63] , wherein X is selected from the group consisting of chloride, fluoride, nitric acid, and bicarbonate.

[65] The composition according to any one of the above

[63] to

[64] , wherein the choline-containing solution comprises choline chloride, phosphorylcholine, or both.

[66] The composition according to any one of the above

[56] to

[65] , wherein the solubilating buffer further comprises an antifoaming agent.

[67] The composition according to any one of the above

[56] to

[66] , wherein the solubilating buffer does not contain an antifoaming agent.

[68] The composition according to any one of the above

[56] to

[67] , wherein the lysis buffer further comprises at least one thiol, which may comprise L-cysteine ​​HCl, sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid, mercaptoethanol, mercaptoethanesulfonic acid, thioglycerol, or any combination thereof, and which may further comprise the concentration of the at least one thiol in the lysis buffer at about 0.005 g / L to 4 g / L.

[69] The composition according to

[68] , wherein the at least one thiol comprises L-cysteine ​​in the lysis buffer at a concentration of about 0.01 g / L to about 2.5 g / L, and / or sodium thioglycolate in the lysis buffer at a concentration of about 0.01 g / L to about 2.5 g / L.

[70] The composition according to any one of the above

[56] to

[69] , wherein the solubilating buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilating buffer is about 0.01 g / L to about 80 g / L.

[71] The composition according to any one of the above

[56] to

[70] , wherein the lysis buffer further comprises a nutritional base solution containing one or more of the following in the lysis buffer: casein peptone at a concentration of about 8 g / L to about 35 g / L, sodium chloride at a concentration of about 2 g / L to about 10 g / L, soy peptone at a concentration of about 1.5 g / L to about 15 g / L, and potassium phosphate at a concentration of about 0.5 g / L to about 5 g / L, and at least one other nutrient.

[72] The composition according to

[71] , wherein the nutrient medium contains at least one other nutrient in the lysis buffer at a concentration of about 10 g / L to about 50 g / L.

[73] The above at least one other nutrient is i) tryptone, ii) soy, iii) NaCl, iv) dipotassium phosphate (K 2 Hpo 4 The composition according to any one of the above

[71] to

[72] , comprising a nutrient medium containing one or more types of glucose, (v) (a), and (v) glucose.

[74] The composition according to any one of the above

[56] to

[73] , wherein the lysis buffer further comprises one or more of a nutrient medium, an isotonic buffer, a peptone, and a salt, and the concentration of the nutrient medium in the lysis buffer is optionally about 10 g / L to about 50 g / L, and the nutrient medium may further comprise Triptycase soy medium.

[75] The composition according to

[74] , wherein the isotonic buffer comprises sodium phosphate, potassium phosphate, phosphate-buffered saline, physiological saline, or any combination thereof, and the concentration of the isotonic buffer in the dissolution buffer is sometimes about 1 g / L to about 20 g / L.

[76] The composition according to any one of the above

[56] to

[75] , wherein the peptone comprises casein peptone and / or soy peptone.

[77] The composition according to any one of the above

[56] to

[76] , wherein the solubility buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof.

[78] The composition according to any one of the above

[56] to

[77] , wherein the solubilating buffer further comprises at least one additional nonionic surfactant, and optionally the at least one additional nonionic surfactant comprises a saponin.

[79] The composition according to any one of

[56] to

[77] , wherein the solubilating buffer does not contain an additional nonionic surfactant.

[80] The composition according to any one of the above

[56] to

[79] , wherein the solubilating buffer does not contain a buffering agent.

[81] The composition according to any one of the above

[56] to

[80] , wherein the solubilating buffer is acidic.

[82] The lysis buffer is (a) Saponins; (b) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, non-surfactant sulfobetaine (NDSB 201), amphipol (PMAL-C8), and one or more surfactants selected from the group consisting of methyl-β-cyclodextrin; (c) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether - one or more surfactants selected from this group; (d) Sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C 7 One or more surfactants selected from the group consisting of BzO; (e) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and / or (f) Structure C 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 Polyoxyethylene surfactant containing 9-10 oxyethylene hydrophilic head groups. A composition according to any one of the above

[56] to

[81] , which does not contain [the specified substance].

[83] The composition according to any one of the claims

[56] to

[82] , wherein at least one microorganism remains intact in the presence of the SDA.

[84] The composition according to any one of

[56] to

[83] , wherein the SDA does not damage the at least one microorganism.

Claims

1. A step of producing a processed sample by contacting a sample containing blood cells and at least one microorganism with a lysis buffer, wherein the lysis buffer contains a somatic cell digestive agent (SDA) capable of lysing the blood cells in the sample, and the SDA is given by the following formula 1 【Chemistry 1】 (In the formula, x is an integer from 2 to 20, and y is an integer from 6 to 11) This is a compound of the form, This step dissolves the blood cells in the sample. A method for processing a sample, including the processing of the sample.

2. The method according to claim 1, (a) y is an integer between 8 and 10. (b) x is an integer between 5 and 15. (c) The concentration of SDA in the lysis buffer is 0.01 g / L to 10 g / L. and / or (d) The concentration of the SDA in the lysis buffer is 0.01% (w / w) to 10% (w / w). The above method.

3. The method according to claim 1 or 2, wherein the SDA is nonoxynol-9.

4. A method according to any one of claims 1 to 3, (a) The sample is derived from a blood culture of a subject suspected of being infected. (b) The sample includes a positive blood culture sample which has been determined to contain at least one microorganism. (c) The at least one microorganism is selected from the group including Gram-positive bacteria, Gram-negative bacteria, and yeasts. (d) The at least one microorganism includes one or more of the following: Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pneumoniae. (e) The contact step includes ultrasonic treatment, osmotic shock, chemical treatment, or any combination thereof. and / or (f) The lysis buffer comprises one or more proteinases and / or one or more nucleases. The above method.

5. A method according to any one of claims 1 to 4, (a) comprising the step of isolating at least one microorganism from the treated sample to produce at least one isolated microorganism, (b) The steps include isolating at least one microorganism from the treated sample to produce at least one isolated microorganism, preparing an inoculum from the at least one isolated microorganism, and analyzing the at least one microorganism obtained from the inoculum, (c) Isolating at least one microorganism from the processed sample to produce at least one isolated microorganism, and depositing at least a portion of the pellet containing the at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of the at least one microorganism by mass spectrometry, or The process involves isolating at least one microorganism from the processed sample to produce at least one isolated microorganism, depositing at least a portion of the pellet containing the at least one isolated microorganism onto a surface suitable for placement in an apparatus configured to determine the identity of the at least one microorganism by mass spectrometry, and drying the deposited sample. Steps and The step of treating the deposited sample with a volatile acid solution, wherein the volume percentage of the volatile acid is at least 70% of the volatile acid solution combined with the deposited sample, and / or (d) A step of contacting the sample with the choline-containing solution before, simultaneously with, and / or after contact with the lysis buffer, The choline-containing solution is given by the following formula 2 【Chemistry 2】 (In the formula, R 1 , R 2 , and R 3 (where X independently represents a group selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and X represents the loaded electrolytic group.) The step comprises a quaternary ammonium salt containing an N,N,N-trimethylethanolammonium cation, selected from the group consisting of the following: The above method.

6. The method according to claim 5, In step (a) of claim 5, (i) The step of isolating the at least one microorganism from the treated sample includes the step of separating the at least one microorganism from lysed blood cells, (ii) The step of isolating the at least one microorganism from the treated sample includes the step of separating the at least one microorganism from the lysed blood cells, The steps include: centrifuging the processed sample to produce a pellet and a supernatant; The step of discarding the supernatant while retaining the pellet containing at least one isolated microorganism, and / or (iii) The method further comprises the steps of preparing a plate pure culture from at least one isolated microorganism and analyzing the microorganism obtained from the plate pure culture, The above method.

7. The method according to claim 5, In step (c) of claim 5, (i) The volatile acid solution is an aqueous solution of a volatile acid or a volatile solution in an organic solvent. (ii) The volatile acid solution, when combined with the deposited sample, is a formic acid aqueous solution with a volume percentage of 70% to 90%. (iii) The step of treating the deposited sample with a volatile acid solution includes the step of drying the deposited sample treated with the volatile acid solution, (iv) The step of treating the deposited sample with a volatile acid solution includes the step of placing a matrix on top of the treated deposited sample, (v) The step of treating the deposited sample with a volatile acid solution includes the step of drying the treated deposited sample on which the matrix is ​​arranged, and / or (vi) The process includes treating the deposited sample with an organic solvent and drying the deposited sample treated with the organic solvent before treating the deposited sample with a volatile acid solution. The above method.

8. A method according to claim 5, wherein in step (d) of claim 5, (a) The choline-containing solution comprises choline chloride, phosphorylcholine, or both. (b) When in contact with the sample, the final concentration of choline is 0.25% by volume or 1% by volume or The concentration of choline in the sample during the contact step is in the range of 0.25% by volume to 10% by volume. and / or (c) The contact step comprises incubating the sample with the choline-containing solution for up to 20 minutes, wherein the incubation temperature is room temperature. The above method.

9. A method according to any one of claims 1 to 8, (a) The dissolution buffer further contains an antifoaming agent, or the dissolution buffer does not contain an antifoaming agent. (b) The lysis buffer further comprises at least one thiol, (c) The solubilizing buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilizing buffer is 0.01 g / L to 80 g / L. (d) The lysis buffer further comprises a nutritional base solution containing (d1) (i) casein peptone at a concentration of 8 g / L to 35 g / L in the lysis buffer, (ii) sodium chloride at a concentration of 2 g / L to 10 g / L in the lysis buffer, (iii) soy peptone at a concentration of 1.5 g / L to 15 g / L in the lysis buffer, and (iv) potassium phosphate at a concentration of 0.5 g / L to 5 g / L in the lysis buffer, and (d2) at least one other nutrient. (e) The lysis buffer further comprises one or more of the following: nutrient medium, isotonic buffer, peptone, and salt. (f) The lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof. and / or (g) The lysis buffer further comprises at least one additional nonionic surfactant, the lysis buffer further comprises a saponin, or the lysis buffer does not contain the additional nonionic surfactant. The above method.

10. A method according to any one of claims 1 to 9, (a) The method further comprises the step of identifying the at least one microorganism, (b) The SDA does not harm the at least one microorganism. (c) The at least one of the microorganisms remains intact in the presence of the SDA. (d) The method yields a MALDI score at least 5% higher than the comparative method using a lysis buffer that does not contain the SDA. (e) The lysis buffer selectively dissolves at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the blood cells in the sample. (f) The ratio of lysed blood cells to lysed cells of at least one microorganism after the contact step is at least 2:

1. (g) After the contact step, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells of the at least one microorganism remain intact and / or viable. (h) The lysis buffer does not contain a buffering agent. (i) The solubilizing buffer is acidic. (j) The above method does not include density gradient centrifugation. and / or (k) The lysis buffer is (i) Saponins; (ii) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, Octyl β-D-Glucopyranoside, Saponin, Nonaethylene glycol monododecyl ether (C12E9, polidocenol), Sodium dodecyl sulfate, N-Lauryl sarcosine, Sodium deoxycholate, Bile salt, Hexadecyltrimethylammonium bromide, SB3-10, SB3-12, Amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, Non-surfactant sulfobetaine (NDSB) One or more surfactants selected from the group consisting of 201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin; (iii) One or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers; (iv) Sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C 7 One or more surfactants selected from the group consisting of BzO; (v) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and / or (vi) Structure C 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 Polyoxyethylene surfactant containing 9 to 10 oxyethylene hydrophilic head groups. does not include, The above method.

11. The mass spectrometry in step (c) of claim 5 is electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, ESI-MS / (MS) n , matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS) n , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS) n , quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, and n is an integer greater than zero, The method according to any one of claims 5 to 10

12. A lysis buffer containing a somatic cell digestive agent (SDA) capable of lysing blood cells, wherein the SDA is defined by the following formula 1 【Transformation 3】 A lysis buffer is a compound of the form (where x is an integer from 2 to 20, and y is an integer from 6 to 11), Blood cells and / or their debris, At least one type of microorganism and A composition containing the following:

13. The composition according to claim 12, (a) y is an integer between 8 and 10. (b) x is an integer between 5 and 15. (c) The concentration of SDA in the lysis buffer is 0.01 g / L to 10 g / L. and / or (d) The concentration of the SDA in the lysis buffer is 0.01% (w / w) to 10% (w / w). The above composition.

14. The composition according to claim 12 or 13, wherein the SDA is nonoxynol-9.

15. A composition according to any one of claims 12 to 14, (a) The lysis buffer comprises one or more proteinases and / or one or more nucleases. (b) The composition further comprises a choline-containing solution comprising at least one quaternary ammonium salt comprising an N,N,N-trimethylethanolammonium cation selected from the group consisting of the following formula 2, 【Chemistry 4】 In the formula, R 1 , R 2 , and R 3 This independently represents a group selected from the group consisting of saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic groups, and combinations thereof, and (i) X represents the load base, (ii) X is selected from the group consisting of chlorides, fluorides, nitric acid, and bicarbonate. and / or (iii) The choline-containing solution comprises choline chloride, phosphorylcholine, or both. (c) The solubilating buffer further contains an antifoaming agent, or the solubilating buffer does not contain an antifoaming agent. (d) The lysis buffer further comprises at least one thiol, (e) The solubilizing buffer further contains ammonium chloride, and the concentration of ammonium chloride in the solubilizing buffer is 0.01 g / L to 80 g / L. (f) The lysis buffer further comprises a nutritional base solution containing (f1) one or more of the following: (i) casein peptone in the lysis buffer at a concentration of 8 g / L to 35 g / L, (ii) sodium chloride in the lysis buffer at a concentration of 2 g / L to 10 g / L, (iii) soy peptone in the lysis buffer at a concentration of 1.5 g / L to 15 g / L, and (iv) potassium phosphate in the lysis buffer at a concentration of 0.5 g / L to 5 g / L, and (f2) at least one other nutrient. (g) The lysis buffer further comprises one or more of the following: nutrient medium, isotonic buffer, peptone, and salt. (h) The lysis buffer further comprises sodium pyruvate, yeast extract, sodium citrate, meat peptone, dextrose, phosphate buffered saline, or any combination thereof. (i) The lysis buffer further comprises at least one additional nonionic surfactant, the lysis buffer comprises a saponin, or the lysis buffer does not contain the additional nonionic surfactant. (j) The lysis buffer does not contain a buffering agent. (k) The solubilizing buffer is acidic. (l) The solubilizing buffer is (i) Saponins; (ii) Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630, Arlasolve® 200, Brij® 96 / 97, CHAPS, Octyl β-D-Glucopyranoside, Saponin, Nonaethylene glycol monododecyl ether (C12E9, polidocenol), Sodium dodecyl sulfate, N-Lauryl sarcosine, Sodium deoxycholate, Bile salt, Hexadecyltrimethylammonium bromide, SB3-10, SB3-12, Amidosulfobetaine-14, C7BzO, Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84, Non-surfactant sulfobetaine (NDSB) One or more surfactants selected from the group consisting of 201), Amphipol (PMAL-C8), and methyl-β-cyclodextrin; (iii) One or more surfactants selected from the group consisting of Triton® X-100, Triton® X-100-R, Triton® X-114, NP-40, Igepal CA 630, Arlasolve 200, Brij® 96 / 97, CHAPS, octyl β-D-glucopyranoside, saponins, and nonaethylene glycol monododecyl ethers; (iv) Sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C 7 One or more surfactants selected from the group consisting of BzO; (v) One or more surfactants selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, and polidocenol; and / or (vi) Structure C 12-18 / E 9-10 (In the formula, C 12-18 This represents the carbon chain length of 12 to 18 carbon atoms, E 9-10 Polyoxyethylene surfactant containing 9 to 10 oxyethylene hydrophilic head groups. does not include, (m) The at least one of the microorganisms remains intact in the presence of the SDA. and / or (n) The SDA does not damage the at least one type of microorganism. The above composition.

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