WO2018062573A1

WO2018062573A1 - Method for recovering cells

Info

Publication number: WO2018062573A1
Application number: PCT/JP2017/036399
Authority: WO
Inventors: 保孝幸; 森　安義
Original assignee: 栄研化学株式会社
Priority date: 2016-09-29
Filing date: 2017-09-29
Publication date: 2018-04-05

Abstract

The purpose of the present invention is to provide an efficient method for recovering cells such as microorganisms or animal cells; specifically, the present invention pertains to a method for recovering cells, said method including a step for mixing cells and magnetic particles in the presence of an alcohol.

Description

Cell recovery method

The present invention relates to a method for recovering microbial cells or animal cells in a specimen, for example.

Genetic detection kits for microbial cells or animal cells such as pathogens have minimal detection sensitivity. Usually, the amount of specimen used in the genetic test is several to several tens of μL per test. For this reason, for example, a sample containing 1,000 or more pathogens per mL of sample can be determined as positive, but a sample containing less than 1,000 pathogens cannot be determined as positive and cannot be detected.
As a means for such a problem of detection sensitivity, a method of increasing the sample amount of the kit can be mentioned. However, simply increasing the amount of the sample simultaneously dilutes the reagent component and also increases the amount of the inhibitory component derived from the sample, which may cause erroneous determination in the test result. In order to avoid this problem, an operation for separating and concentrating microbial cells or animal cells such as pathogens from the sample components is necessary.
Usually, although the centrifugation method is mentioned as a method for separating and concentrating microbial cells or animal cells, the centrifugation method requires expensive equipment and a use environment.
Thus, in order to improve the detection performance of the genetic test kit for microbial cells or animal cells such as pathogens, an inexpensive and simple method for concentrating microbial cells or animal cells is desired.
As a conventionally known method for recovering cells, Patent Document 1 discloses an aliphatic solution at a concentration in which only bacterial cells are aggregated and high molecular polysaccharides are not aggregated in a culture broth of bacteria that secrete and produce polymeric polysaccharides outside the bacterial cells. Alcohol is added to agglomerate cells to separate the cells, while alcohol is added to the culture broth from which the cells are removed to precipitate and recover the polymer polysaccharide. A purification method is disclosed.
Patent Document 2 discloses a microorganism recovery method in which microparticles in a sample are adsorbed to the microparticles by bringing the microparticles into contact with the sample.
In Patent Document 3, a slurry liquid is prepared by contacting water containing a microorganism and a magnetic substance or an aggregate thereof, and the slurry liquid is solid-liquid separated into an aggregate cake and water by filtering the slurry liquid. Disclosed is a method for concentrating microorganisms, in which the cake is separated into microorganisms and magnetic substances to concentrate the microorganisms.
However, conventionally, there has not been known a method capable of efficiently concentrating microbial cells or animal cells by using magnetic particles and alcohol in combination.

JP-A-6-209783 International Publication No. 2006/123781 JP 2012-187083 A

As described above, an efficient method for recovering microbial cells or animal cells is desired in order to improve the detection performance of genetic test kits for microbial cells or animal cells such as pathogens.
Then, an object of this invention is to provide the efficient collection | recovery method of microbial cells, such as bacteria, and an animal cell.
As a result of diligent research to solve the above problems, it was found that microbial cells or animal cells co-aggregate with the magnetic particles in the presence of magnetic particles and alcohol, and the microbial cells or animal cells can be easily recovered. The invention has been completed.
That is, the present invention includes the following.
(1) A method for recovering cells, comprising a step of mixing cells and magnetic particles in the presence of alcohol.
(2) The method according to (1), wherein the cell is a microbial cell or an animal cell.
(3) The method according to (1) or (2), wherein the magnetic particles are magnetic particles containing magnetite and / or silica as a surface material.
(4) Any of (1) to (3), wherein the alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol, ethylene glycol, 2,2′-iminodiethanol, polyethylene glycol, and mixtures thereof. The method according to claim 1.
(5) The method according to (4), wherein the polyethylene glycol has a molecular weight of 20,000 or less.
(6) A test method including the cell recovery method according to any one of (1) to (5).
(7) A cell recovery kit containing magnetic particles and alcohol.
(8) The kit according to (7), wherein the cells are microbial cells or animal cells.
(9) The kit according to (7) or (8), wherein the magnetic particles are magnetic particles containing magnetite and / or silica as a surface material.
(10) Any of (7) to (9), wherein the alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol, ethylene glycol, 2,2′-iminodiethanol, polyethylene glycol, and mixtures thereof. A kit according to claim 1.
(11) The kit according to (10), wherein the polyethylene glycol has a molecular weight of 20,000 or less.
(12) A test kit comprising the cell recovery kit according to any one of (7) to (11).
This specification includes the disclosure of Japanese Patent Application No. 2016-192190, which is the basis of the priority of the present application.

It is a photograph which shows the observation result (x200) of the magnetite particle | grains in the various solvents in Example 1 (left: DW, right: 70% EtOH). It is a photograph which shows the observation result after mixing a magnetite particle | grain with the fungus | bacteria liquid in Example 1, and making it acid-acid-bacteria dyeing | aggregating on EtOH addition conditions (photograph a): * 200, photograph b), and c). : Observed at x400). Photo a): shows the state of co-aggregation of magnetite particles and bacterial cells. The black part shows magnetite particles, and the part dyed red shows microbial cells. Photo b): shows the appearance of mycobacteria in a typical bacterial cell shape (gonococcal shape) stained red in the aggregate of magnetite particles. Photo c): shows the mycobacteria cells in the field of view with few magnetite particles and the magnetite particles adsorbed so as to border the cells. The basic microbial cell collection | recovery protocol in the method of this invention is shown. By the method of the present invention, the bacterial cells in the bacterial solution or specimen are recovered as an aggregate consisting of magnetite particles and bacterial cells. The collected agglomerates can then be subjected to an appropriate nucleic acid extraction method or genetic test.

The cell collection method according to the present invention (hereinafter referred to as “the present method”) is a method including a step of mixing cells and magnetic particles in the presence of alcohol. According to the present invention, by mixing cells and magnetic particles in the presence of alcohol, the cells and magnetic particles co-aggregate to form aggregates. The aggregate formed in this way can be collected and used as an object for a test such as a genetic test.
Cells collected by this method are present in, for example, specimens (for example, clinical specimens such as sputum, gastric juice, stool emulsion, urine, pleural effusion, whole blood, serum, plasma). Examples of the cells include animal cells such as humans and cells such as microorganisms. Further, the microorganism may be a microorganism (or pathogen) belonging to any genus, such as Mycobacterium bovis BCG strain, Mycobacterium tuberculosis (Mycobacterium tuberculosis), Gram-positive bacteria such as mycobacteria, including Mycobacterium avium and Mycobacterium intracellulare, Bordetella pertussis li, Escherichia coli, etc. Gram-negative bacteria, yeasts (Saccharomyces cerevisiae) and other fungi.
The magnetic particles used in this method may be any particles as long as they have magnetism, and examples thereof include particles containing (or consisting of) magnetite and / or silica as surface materials. In other words, in this method, magnetic particles coated with magnetite and / or silica can be used. In addition, the magnetic particles may be surface-modified with a functional group such as a carboxyl group, a methyl group, an octadecyl group, albumin, or boronic acid. Furthermore, the shape of the magnetic particle is not particularly limited, and examples thereof include a spherical shape and an octahedron. Further, examples of the size of the magnetic particles include a diameter (φ) of 25 nm to 2 μm. As these magnetic particles, commercially available particles can be used.
On the other hand, examples of alcohol used in the present method include methanol, ethanol (EtOH), 1-propanol, isopropyl alcohol (2-propanol, IPA), ethylene glycol, 2,2′-iminodiethanol, polyethylene glycol (PEG), and These mixtures etc. are mentioned. Examples of PEG include those having a molecular weight of 20,000 or less (monoethylene glycol to molecular weight 20,000).
In this method, first, magnetic particles and alcohol are added to a specimen containing cells to be collected and mixed. The magnetic particles and alcohol may be added separately to the specimen, or may be added simultaneously. For example, 0.5 to 20 mg of magnetic particles and 1,400 μL of alcohol are added to 600 μL of a specimen containing cells, mixed, and allowed to stand for 0 to 10 minutes. When using EtOH, for example, EtOH is added to a final concentration of 40 to 90%. When IPA is used, IPA is added so that the final concentration is, for example, 20 to 90%. When PEG (molecular weight 1,000) is used, PEG (molecular weight 1,000) is added so that the final concentration is, for example, 2.5 to 30%. When PEG (molecular weight: 8,000) is used, PEG (molecular weight: 8,000) is added so that the final concentration is, for example, 5.25 to 21%.
Next, a magnet is used to collect an agglomerate containing cells and magnetic particles. In this way, cells in the specimen can be collected. Thus concentrated cells (aggregates) can be subjected to tests such as genetic tests. Therefore, the present invention also relates to a testing method such as genetic testing, which includes a step of extracting nucleic acid from cells collected by the method and a step of subjecting the extracted nucleic acid to testing such as genetic testing.
In the present invention, genetic testing refers to PCR (for example, JP-A-2001-286300 and JP-A-62-2000281), SDA (for example, JP-A-5-192195), NASBA (for example, JP-A-5-192181). 2-005864), RCA (Proceedings of the National Academy of Sciences of the United States of America 92: 4641-4645 (1995)), and LAMP (for example, Japanese Patent No. 3313358). And an inspection method using a nucleic acid amplification method such as 2-002934).
As a method for extracting nucleic acid from cells collected by this method, nucleic acid extraction methods generally used for nucleic acid extraction such as genomes and plasmids (for example, phenol / chloroform method and alkali extraction method) and commercially available methods are available. The nucleic acid extraction method using the nucleic acid extraction kit is not particularly limited.
The present invention also relates to a cell collection kit containing magnetic particles and alcohol used in the present method. In addition to magnetic particles and alcohol, the cell collection kit can contain, for example, reagents, containers, instruction manuals, etc. used for cell collection.
Furthermore, the cell collection kit can be included in a test kit for genetic testing or the like, and can also be provided as a kit for a pretreatment reagent. The test kit can further include reagents, containers, instruction manuals, etc. used for nucleic acid extraction and genetic testing.
EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

Observation of magnetite particles Objectives The objectives of this example are as follows:
Observation of magnetite particles agglomerated in alcohol;
Observation of the location of bacteria when the cells are aggregated under the condition where the cells and magnetite particles coexist.
2. Materials The materials used in this example were as follows:
Mycobacterium bovis BCG Tokyo KK-12-21 strain;
Magnetite particles: ferric oxide (III) iron (II) (Wako Pure Chemical Industries);
Physiological saline (hereinafter referred to as “raw food”, Otsuka Pharmaceutical Factory);
Magnet for collecting magnets;
Aggregating solvent: ethanol (hereinafter referred to as “EtOH”; Wako Pure Chemical Industries), distilled water (hereinafter referred to as “DW”; Otsuka Pharmaceutical Factory);
Optical microscope: Olympus biological microscope CH2 (OLYMPUS);
Spectrophotometer: CO8000 Biowave (Biochrom Ltd.);
2. Tielnelsen staining solution: fuchsin carboxylic acid solution, methylene blue solution Method 1) Preparation of Bacterial Solution The cultured Mycobacterium bovis BCG Tokyo KK-12-21 strain 1 platinum ear equivalent was suspended in a raw diet (hereinafter, the cells of the bacterium are referred to as “bacteria”, a saline suspension. Is referred to as “bacterial fluid”).
Subsequently, in order to remove free DNA in the bacterial solution, a washing operation by centrifugation (3,000 × g, 20 min) was performed twice. After washing, 1 mL of saline was added to the centrifugal sediment and resuspended.
Using a spectrophotometer, the absorbance value of the bacterial solution was measured at an absorption wavelength of 600 nm, and McFarland No. 1 (OD600 = 0.25). The prepared bacterial solution was 10 ⁷ CFU / mL, and a 10 ⁶ CFU / mL bacterial solution was prepared by 10-fold dilution.
2) Cell recovery in bacterial solution 2.5 mg of magnetite particles were suspended in 300 μL of saline or 10 ⁶ CFU / mL bacterial solution. Next, 700 μL of aggregation solvent (EtOH or DW) was mixed (final concentration 70%).
After mixing, magnetite particles were collected with a magnet and the supernatant was removed. After removing the supernatant, the magnetite particles were resuspended in 300 μL of raw food.
3) Observation Observation (1): Magnetite particles subjected to the treatment of 2) were observed at a magnification of x200 under the condition of using raw food.
Observation (2): The magnetite particles treated in 2) were subjected to mycobacterial staining under the conditions of using bacterial solution, and then observed at x200 to x400.
4). Results The results are shown in FIGS.
Observation (1): When DW was added, a state where magnetite particles were dispersed without being aggregated was observed. On the other hand, it was observed that the magnetite particles aggregated when EtOH was added (FIG. 1). The phenomenon that the magnetite particles aggregate in the presence of EtOH may have a hydrophobic interaction, but the detailed mechanism is not clear.
Observation (2): Bacteria stained in red in magnetite particles were observed (FIG. 2a) × 200). When observed at x400, in the field of view where the magnetite particles are dense, a typical cell shape of mycobacteria is observed along with the appearance of the cells being buried in the aggregated particles (Fig. 2b) x400). In the rough visual field region, it was observed that the cell surface was covered with adsorbed magnetite particles (FIG. 2c) × 400).
Under the conditions that magnetite particles cause agglomeration phenomenon, it was confirmed that the cell body adsorbs magnetite particles on the surface and co-aggregates with the magnetite particle. It was expected that it could be recovered.

Conditions necessary for cell recovery by this method Purpose The purpose of this example is to examine the conditions necessary for cell recovery by this method.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and cell recovery Mycobacterium bovis BCG Tokyo KK-12-21 strain;
Magnetite particles: ferric oxide (III) iron (II) (Wako Pure Chemical Industries);
Raw food (Otsuka Pharmaceutical Factory);
Magnet for collecting magnets Aggregating solvent: EtOH (Wako Pure Chemical Industries), 2-propanol (hereinafter referred to as “IPA”; Wako Pure Chemical Industries), polyethylene glycol 1,000 (hereinafter referred to as “PEG 1,000”) Wako Pure Chemical Industries), DW (Otsuka Pharmaceutical Factory), and PEG 1,000 were prepared in 20% (w / v) aqueous solution and used in the following experiments. ;
Spectrophotometer: CO8000 Biowave (Biochrom Ltd.).
2) Gene amplification test Loopamp PURE DNA extraction kit (Eiken Chemical, Patent No. 5432891);
Loopamp Mycobacterium tuberculosis group detection reagent kit (Eiken Chemical);
Loopamp real-time turbidity measurement apparatus LA-320c (Eiken Chemical).
3. Method 1) Bacterial Solution Preparation McFarland No. The same method as in Example 1 was used for the method for preparing the bacterial solution 1.
The prepared bacterial solution was made 10 ⁷ CFU / mL and diluted 10,000 and 100,000 times to prepare bacterial solutions of 10 ³ and 10 ² CFU / mL, respectively.
2) Recovery of bacterial cells in bacterial solution (Fig. 3)
2.5 mg of magnetite particles were suspended in 600 μL of 10 ² CFU / mL bacterial solution. Next, 1,400 μL of aggregating solvent was added and mixed. See Table 2 for the final concentration of flocculation solvent.
Magnetite particles were collected from this mixed solution with a magnet, and the supernatant was removed.
3) Extraction of nucleic acid 1) For each concentration of bacterial solution prepared in “Bacterial solution preparation”, a DNA extract was prepared from 60 μL of each using a Loopamp PURE DNA extraction kit.
On the other hand, 10 mL of DW is added to the agglomerates composed of the magnetite particles and bacterial cells recovered by the method 2) to disperse the magnetite, and then the magnetite particles are collected again, and the supernatant is removed. Magnetite particles were resuspended in the raw food, and a DNA extract was prepared from this resuspension 60 μL (total amount) using a Loopamp PURE DNA extraction kit.
4) Gene amplification test A series of tests using the Loopamp tuberculosis group detection reagent kit is hereinafter referred to as “PURE-TB-LAMP”.
Experiment A: Method 1) DNA extracted from each concentration of bacterial solution prepared in “Bacterial solution preparation” was subjected to PURE-TB-LAMP.
Experiment B: Method 2) DNA extracted from the aggregate collected by the method of “recovering cells in bacterial solution” was subjected to PURE-TB-LAMP.
4). Results The results are shown in Tables 1 and 2.
1) As experiment A, the results of triple measurement of DNA extracted from the bacterial solution of 10 ² , 10 ³ CFU / mL of method 1) with PURE-TB-LAMP (Table 1),
10 ² CFU / mL became negative at n = 2 of triplicate measurements;
10 ³ CFU / mL was positively detected from all triplicate measurements.
In this experiment, the minimum concentration of the bacterial solution that stably obtained a positive result for PURE-TB-LAMP used in the gene amplification test was 10 ³ CFU / mL.
2) As experiment B, the results of triple measurement of DNA extracted from the collected aggregates by PURE-TB-LAMP by the method of method 2) and under the conditions a to f of Table 2 (Table 2) ,
-In condition a where magnetite was added and DW was used as the solvent, all triple measurements were negative;
In conditions b, c, and d where magnetite was added and EtOH, IPA, and PEG were used as solvents, all triple measurements were positive;
In condition e in which magnetite was added and no solvent was used, n = 2 in triplicate measurements were negative;
-Under the condition f where no magnetite was added and EtOH was used as a solvent, all the triple measurements were negative.
As a result of carrying out the cell recovery from 600 μL of the 10 ² CFU / mL bacterial solution, a positive result was stably obtained under the conditions b, c and d of Experiment B.
-The bacterial cells in the bacterial solution tested under the above conditions are collected, and the concentration of the collected bacterial cells (that is, the resuspension bacteria) is equivalent to 10 ³ CFU / mL bacterial solution. thing,
-DNA can be extracted from the resuspension obtained by this method, and nucleic acid amplification reaction can be performed using the obtained DNA as a template,
Therefore, it was determined that the cells were collected by this method.
In addition, from the results of Experiments B and A, the magnetite particles used do not have the ability to recover bacteria, and from the results of Experiment B, conditions f cannot be recovered by using a flocculating solvent alone. Therefore, it was determined that the combination of particles (magnetite particles) and aggregating solvent (EtOH, IPA, PEG) was essential as a condition for cell recovery by this method.

Bacteria recovery by this method (1)
1. Purpose The purpose of this example is to estimate the cell recovery rate by this method.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 2 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Gene amplification test The same material as in Example 2 was used.
3. Method 1) Bacterial Solution Preparation McFarland No. The same method as in Example 1 was used for the method for preparing the bacterial solution 1.
The prepared bacterial solution was made 10 ⁷ CFU / mL and diluted 10,000 times to prepare a 10 ³ CFU / mL bacterial solution.
Subsequently, a 2-fold serial dilution series of 15.6 to 10 ³ CFU / mL was prepared.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 3.
As a result of subjecting DNA extracted from the serial dilution series of the bacterial solution to PURE-TB-LAMP, the minimum detection sensitivity was 500 CFU / mL.
As a result of subjecting the DNA extracted from the aggregate collected using the “recovering bacterial cells in bacterial solution” method of Example 2 to PURE-TB-LAMP, the minimum detection sensitivity was 62.5 CFU / mL.
Compared with the measurement result of the bacterial liquid, the minimum concentration of the bacterial liquid that can stably obtain a positive result has been improved by 8 times, and the amount of the bacterial liquid used for the method of collecting bacterial cells has been increased by 10 times. Considering that there is a difference, it was speculated that this method can recover the cells in the bacterial solution in a considerable proportion.

Bacteria recovery by this method (2)
1. [Purpose] The purpose of this example is to collect cells from a high-concentration bacterial solution and to investigate the recovery rate.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Gene amplification test PURE DNA extraction kit (Eiken Chemical);
Commercial real-time PCR measurement apparatus: Mx3005P (Agilent Technologies);
Template quantification LAMP reaction reagent prepared in house;
It was used.
3. Method 1) Preparation of Bacterial Solution The same method as in Example 1 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test Experiment A: Method 1) The amount of DNA extracted from the bacterial solution prepared in “Bacterial solution preparation” was quantified.
Experiment B: Method 2) The amount of DNA extracted from the agglomerates collected in “Recovering cells in bacterial solution” was quantified.
4). Results The results are shown in Table 4.
Since the quantitative value of Experiment A was (1.6 ± 0.2) × 10 ⁶ copies / mL and the quantitative value of Experiment B was (1.7 ± 0.2) × 10 ⁷ copies / mL, In experiments, it was confirmed that this method can recover almost 100% of the cells in the suspension. Moreover, based on the result of Example 3, it was guessed that this method is applicable to the bacterial solution of a wide concentration range.

DNA recovery by this method Purpose The purpose of this example is to confirm the ability of DNA recovery by this method.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Commercial DNA extraction kit ISOPLANT II (Nippon Gene);
10 mM Tris-HCl (pH 8.0).
3) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Extraction and purification of bacterial genome DNA The cultured Mycobacterium bovis BCG Tokyo KK-12-21 strain 1 platinum ear equivalent was suspended in a raw diet. Subsequently, genomic DNA was extracted and purified with a commercially available DNA extraction kit ISOPLANT II.
The obtained DNA solution was dissolved in 10 mM Tris-HCl (pH 8.0), the absorbance value of the DNA solution was measured at an absorption wavelength of 260 nm using a spectrophotometer, and the copy number was calculated from the obtained weight concentration. did.
According to the calculated copy number, a 10-fold serial dilution series DNA solution of 100 to 10 ⁵ copies / mL was prepared by saline.
2) DNA recovery from DNA solution The DNA solution prepared in Method 1) was subjected to the same method as in Example 2. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test A series of tests using the Loopamp tuberculosis group detection reagent kit is hereinafter referred to as “PURE-TB-LAMP”.
Experiment A: DNA extracted from each concentration of DNA solution prepared in Method 1) was subjected to PURE-TB-LAMP.
Experiment B: DNA extracted from the aggregate collected in method 2) was subjected to PURE-TB-LAMP.
4). Results The results are shown in Table 5.
In experiment A, all positive reactions were detected up to 10 ³ copy / mL, and in experiment B, all positive reactions were detected up to 10 ⁴ copy / mL. Therefore, it was confirmed that this method hardly recovered DNA itself. The results of Examples 2 to 4 suggested that the detection sensitivity of the genetic test was increased by recovering the cells by this method.

Examination of available magnetic particle types Purpose The purpose of this example is to investigate the relationship between the surface material of magnetic particles, the presence / absence of functional groups on the particle surface, the shape and particle size, and the recovery of bacterial cells.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
For the magnetic particle types used in this experiment, see Table 6. As magnetite particles whose surfaces were coated with silica, SiMAG-active, SiMAG-hydrophobe, and SiMAG-affinity series (Chemicell) were used. Magnetite particles include ferric oxide (III) iron (II) (Wako Pure Chemical Industries), ferric oxide (III) iron (II) nanoparticles (Wako Pure Chemical Industries), and magnetite product series (Mitsui). Metal Industry Co., Ltd.) was used.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 6.
As a result of triple measurement of 10 ² CFU / mL of the bacterial solution of 60 2 in PURE-TB-LAMP, n = 2 in the triple measurement was negative.
From 12 types of each of the particles from which the cells were collected, all obtained positive results in triplicate measurement.
From these results, it was confirmed that the cells can be recovered by this method regardless of the surface material of the particles, the presence / absence of the surface-modifying functional group, the shape and the particle size within the range examined in this experiment.

1. Examination of the amount of magnetic particles used Purpose The purpose of this example is to investigate the range of usable particle amounts.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
Refer to Table 7 for the amount of particles used.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 7.
Bacteria could be collected in the range of 0.5 to 20 mg of particles added.

Examination of coagulation solvent (1)
1. Purpose The purpose of this example is to investigate available solvent species (alcohol species).
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used.
Refer to Table 8 for the agglomerated solvent species used.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. All flocculating solvents were used at a final concentration of 70%.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 8.
In addition to EtOH and IPA (2-propanol), bacterial recovery was observed with methanol, 1-propanol, ethylene glycol, and 2,2′-iminodiethanol.

Examination of coagulation solvent (2)
1. Purpose The purpose of this example is to investigate the optimum concentration range of alcohol as a coagulation solvent species.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. EtOH and IPA were used as the aggregation solvent.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The amount of the aggregation solvent added to 600 μL of the bacterial solution was changed. See Table 9 for the concentration of the flocculating solvent at the time of mixing, that is, the range of the final concentration.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 9.
Bacterial recovery was observed in the range of 40 to 80% for EtOH and 20 to 80% for IPA.
From these results, it became clear that there is an optimum concentration range for cell recovery for each aggregation solvent.
The reason that IPA showed a wider effective concentration than EtOH was considered to be due to the strength of hydrophobicity (difference in the length of the carbon chain of the alkyl group exhibiting hydrophobicity).

Examination of coagulation solvent (3)
1. Purpose The purpose of this example is to investigate the molecular weight and optimal concentration range of PEG.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used.
The aggregating solvents used were four kinds of PEG molecular weights of 1,000, 3,350,8,000, and 20,000, and 20% (w / v) aqueous solutions were prepared and used in Experiment A. Further, a 50% (w / v) aqueous solution of PEG 1,000 and a 30% (w / v) aqueous solution of PEG 8,000 were prepared and used in Experiment B.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Bacterial cell recovery in bacterial solution Using the same method as in Example 2, the following two types of experiments were conducted.
Experiment A: The condition of bacterial recovery with PEG 1,000 to 20,000 was investigated under the condition of a final concentration of 15%.
Experiment B: The microbial recovery state was investigated by changing the amount of PEG 1,000 and PEG 8,000 added to 600 μL of the bacterial solution.
See Tables 10 to 12 for the concentration of the flocculating solvent used in Experiments A and B, that is, the range of final concentrations.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Tables 10-12.
From the results of this experiment A, bacterial recovery was observed at all molecular weights used.
From the results of Experiment B, bacterial recovery was observed in the range of 2.5 to 30% for PEG 1,000 and 5.25 to 21% for PEG 8,000.
Based on the results of this experiment and the ethylene glycol (monomer) of Example 8, it was confirmed that the molecular weight can be recovered in the range of 62.1 to 20,000.
In addition, it has been clarified that there is an optimum concentration range for cell recovery even in the case of PEG.

Recovery of bacterial cells (1)
1. Purpose The purpose of this example is the application of the present method to the same species of bacteria other than Mycobacterium bovis.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
In this experiment, Mycobacterium tuberculosis H37Rv KK11-291 strain was used.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 3 was used. However, in this experiment, a 2-fold serial dilution series of 7.8 to 500 CFU / mL was prepared.
2) Bacterial cell recovery in bacterial solution The same method as in Example 3 was used. The final concentration of the aggregation solvent EtOH is 70%.
3) Extraction of nucleic acid The same method as in Example 3 was used.
4) Gene amplification test The same method as in Example 3 was used.
4). Results The results are shown in Table 13.
Experiment A: Method 1) The DNA extracted from each dilution of the bacterial solution prepared in “Bacterial solution preparation” was subjected to PURE-TB-LAMP, and the minimum detection sensitivity was examined. As a result, it was 125 CFU / mL.
Experiment B: Method 2) As a result of subjecting DNA extracted from the aggregate collected by the method of “recovering bacterial cells in bacterial solution” to PURE-TB-LAMP, the minimum detection sensitivity was 15.6 CFU / mL.
From these results, it was confirmed that this method can be applied to other species of the genus Mycobacterium other than Mycobacterium bovis.
In Experiment A, the detection sensitivity of Mycobacterium tuberculosis used in this experiment was 8 times higher than that of Mycobacterium bovis. (IS6110) copy number difference (16 for Mycobacterium tuberculosis H37Rv KK11-291 and 2 for Mycobacterium bovis BCG Tokyo KK-12-21) It was.

Recovery of bacterial cells (2)
1. Purpose The purpose of this example is to investigate the range of applicable bacterial species 2: gram-negative bacteria.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 2 were used.
In this experiment,
Bordetella pertussis Tohama strain;
Magnetite particles;
Aggregation solvent: Polyethylene glycol 8000 (PEG 8,000, Wako Pure Chemical Industries) and PEG 8,000 were prepared in a 15% (w / v) aqueous solution and used in the following experiments.
It was used.
2) Gene amplification test Loopamp SR DNA extraction kit (Eiken Chemical);
Loopamp pertussis group detection reagent kit (Eiken Chemical);
Loopamp real-time turbidity measurement device LA-320C (Eiken Chemical);
Tabletop centrifuge: Microcooled centrifuge 3780 (Kubota Corporation);
It was used.
3. Method 1) Preparation of Bacterial Solution Cultured Bordetella pertussis 1 platinum ear equivalent was suspended in saline (hereinafter, the cells of the bacteria are referred to as “bacteria” and the saline suspension as “bacterial fluid”).
Subsequently, in order to remove free DNA in the bacterial solution, a washing operation with centrifugation (15,000 × g, 20 min) was performed twice. After washing, 1 mL of saline was added to the centrifugal sediment and resuspended.
Using a spectrophotometer, the absorbance value of the bacterial solution was measured at an absorption wavelength of 600 nm, and McFarland No. It was adjusted to 0.5 (OD600 = 0.13). The prepared bacterial solution is 1.5 × 10 ⁸ CFU / mL, diluted 10,000 times to prepare a 1.5 × 10 ³ CFU / mL bacterial solution, and subsequently diluted 4 to 8 times. 3,750, 1,875 CFU / mL of bacterial solution was prepared.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. However, 300 μL was used as the bacterial cell recovery bacterial solution, and 700 μL was used as the aggregation solvent (final concentration of the aggregation solvent was 10.5%).
3) Extraction of nucleic acid The same method as in Example 2 was used. However, the Loopamp SR DNA extraction kit was used as the DNA extraction kit. In addition, the agglomerates collected in method 2) were directly resuspended in 50 μL of raw food without using a washing step with DW, and used for the DNA extraction kit.
4) Bacteria recovery by centrifugation 300 μL of each concentration of bacterial solution was subjected to centrifugation under conditions of 15,000 × g and 5 min. The supernatant was then removed and resuspended in 50 μL of saline.
5) Gene Amplification Test Using 10 μL of DNA extracted from the bacterial solution or the collected aggregate in Method 3), quadruplicate measurement was performed using a Loopamp Pertussis group detection reagent kit.
4). Results The results are shown in Table 14.
As a result of measuring each DNA extract with the Loopamp Pertussis group detection reagent kit, both samples were positive only in the case of using PEG 8,000, and all samples were positive in the case of centrifuging. The positive detection rate was 1/4 at 3/4 and 1,875 CFU / mL.

Recovery of bacterial cells (3)
1. Purpose The purpose of this example is to investigate the range of applicable bacterial species.
2. Material 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 2 were used.
In addition, as the bacterial species used in this experiment,
-Mycobacterium bovis BCG Tokyo KK12-21 strain-Mycobacterium avium JATA51-01 strain-Mycobacterium intracellularis E. coli ) ATCC11775 strain-About 1 platinum loop of yeast (Saccharomyces cerevisiae) BY611 strain was collected from the medium and suspended in a saline solution.
Further, EtOH and PEG 1,000 were used as the aggregation solvent. For PEG 1,000, a 15% (w / v) aqueous solution was prepared and used in the following experiments.
2) DNA extraction A Loopamp PURE DNA extraction kit (Eiken Chemical) was used.
3) DNA measurement SpectraMax Plus 384 (Molecular Devices) was used for the absorbance measurement.
3. Method 1) Bacterial solution preparation The cultured bacterial cell 1 equivalent of platinum ear was suspended in a raw food.
-In order to remove free DNA in the bacterial solution, washing with centrifugation (15,000 xg, 20 min) was performed twice.
-1 mL of saline was added to the centrifugal sediment and resuspended.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentrations of the aggregation solvent EtOH (70%) and PEG 1,000 are 70% and 10.5%, respectively.
3) Extraction of nucleic acid The following treatment was performed using various bacterial solutions.
Treatment A: Method 1) DNA was extracted from 60 μL of the bacterial solution prepared by “Preparation of bacterial solution” using the method of Example 2.
Process B: Method 2) “Recovering bacterial cells in bacterial solution” method, and collecting aggregates collected using EtOH (70%) as an agglutination solvent. Extracted.
Process C: Method 2) “Recovering bacterial cells in bacterial solution” method, and using PEG 1,000 (15%) as an aggregating solvent to collect and recover the aggregated mass using the nucleic acid extraction method of Example 2 Extracted.
4) DNA concentration measurement Each DNA extract thus obtained was diluted 2 to 10 times with Tris-HCl pH 8.0 (Wako Pure Chemical Industries, Ltd.), and the DNA concentration was calculated from the absorbance measurement value.
4). Results The results are shown in Table 15.
When all the bacterial species used in this test were subjected to the cell collection operation (treatments B and C) according to this method, any DNA extract was prepared (treatment A) without using this method. Compared with the DNA extract, it showed higher DNA concentration and concentration ratio.
From this, it was confirmed that this method can collect cells of acid-fast bacteria (gram-positive bacteria), gram-negative bacteria, and fungi.

Application to various specimen types [Purpose] The purpose of this example is to investigate biological specimen species that can be recovered by this method.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Specimen Sputum, gastric juice, 10% stool suspension supernatant (stool emulsion), urine, pleural effusion, and whole blood were used as biological specimens.
The sputum sample used was a sample obtained by adding 1,200 μL of 0.5 M NaOH to 600 μL of the sample, mixing and dissolving.
3) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used. However, in this operation, a 10 ³ CFU / mL bacterial solution was prepared.
2) Sample preparation 1/10 amount of the bacterial solution of 1) was added to each biological sample to prepare various bacterial solutions of 10 ² CFU / mL.
3) Cell recovery in bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
4) Extraction of nucleic acid The same method as in Example 2 was used.
5) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 16.
All samples listed above were positively detected by LAMP.
From this, it was confirmed that the cells can be recovered from a wide variety of specimen species by this method.

Examination of order of addition of particles and coagulation solvent Purpose The purpose of this example is to investigate the order of addition of particles and flocculating solvent.
2. Materials The materials used in this example were as follows:
1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation 2) Cell recovery in bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%. The addition order of the magnetite particles and the solvent is shown in Table 17.

3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 18.
Experiment A: The positive detection rate was 1/3 in triplicate measurement.
Experiment B: A positive reaction was obtained for all the addition orders of the conditions (1) to (3).
From these results, it was confirmed that this method can be carried out regardless of the order of addition of reagents.

1. Examination of standing time after addition of flocculating solvent Objective Investigation of the relationship between the standing time after addition of the flocculating solvent and cell recovery. Material 1) Bacterial solution preparation and bacterial cell collection The same materials as in Example 3 were used. In this experiment, EtOH was used as the aggregation solvent.
2) Gene amplification test The same material as in Example 3 was used.
3. Method 1) Bacterial solution preparation The same method as in Example 2 was used.
2) Cell recovery in the bacterial solution The same method as in Example 2 was used. The final concentration of the aggregation solvent EtOH is 70%.
The standing time after addition of the aggregating solvent was set to 0, 1, 3, 5, and 10 minutes.
3) Extraction of nucleic acid The same method as in Example 2 was used.
4) Gene amplification test The same method as in Example 2 was used.
4). Results The results are shown in Table 19.
As a result of triple measurement of 10 ² CFU / mL of the bacterial solution of 60 2 in PURE-TB-LAMP, n = 2 in the triple measurement was negative.
When the bacterial cell collection operation according to this method was performed, all the samples were positive in triplicate measurement regardless of the standing time of 0 to 10 minutes.
From these results, it was confirmed that in the range of the standing time examined by this experiment, the present method can recover the cells regardless of the standing time after adding the aggregation solvent to the bacterial solution.

Collection of animal cells Objectives Range survey of applicable cell types: Application of this method to animal cells.
2. Materials The materials used in this example were as follows:
1) Cell suspension preparation, cell recovery Cells: Human peripheral blood mononuclear cells (Kurabo)
Saline (raw food): Otsuka raw food injection (Otsuka Pharmaceutical Factory)
Hemacytometer (Tokyo Glass Instruments)
Magnetite particles: MG-1310 (Mitsui Metals Mining)
Magnet for collecting magnets Aggregating solvent: 20% polyethylene glycol 1,000 (PEG 1,000, Wako Pure Chemical Industries), 20% polyethylene glycol 8,000 (PEG 8,000, Wako Pure Chemical Industries)
2) Gene amplification test Loopamp PURE DNA extraction kit (Eiken Chemical)
Loopamp DNA amplification reagent D (Eiken Chemical)
LAMP primer for detecting human cytochrome gene (CYP2C19) Loopamp real-time turbidity measurement device LA-320c (Eiken Chemical)
3. Method 1) Cell suspension preparation After suspending human peripheral blood mononuclear cells in physiological saline, the cells were counted with a hemocytometer to obtain a cell suspension of 14,300 cells / μL. This was diluted 10 times with physiological saline to prepare a cell suspension having the following concentration.
a. 1,430 cells / μL
b. 143 cells / μL
c. 14.3 cells / μL
2) Cell recovery in suspension The same method as in Example 2 was used.
-2.5 mg of magnetite particles were suspended in 300 μL of cell suspension b.
-700 μL of each aggregation solvent was mixed (final concentration of each PEG = 14%).
-Magnetite particles were collected with a magnet and the supernatant was removed.
-The magnet was removed and 10 mL of physiological saline was added to disperse the magnetite particles.
-The magnetite particles were collected again and the supernatant was removed.
• The magnetite particles were resuspended in 60 μL of physiological saline.
3) Gene amplification test Basically, the same DNA extraction method as in Example 2 was used.
A. Method 1) Using 30 μL of each of the cell suspensions a to c prepared in “Preparation of cell suspension”, DNA extraction was performed with a Loopamp PURE DNA extraction kit.
B. Method 2) DNA extraction was performed with the Loopamp PURE DNA extraction kit using 60 μL of the magnetite particle suspension prepared in “Recovering cells in suspension”.
The DNA extract obtained by the operations of A and B was subjected to a LAMP reaction using Loopamp DNA amplification reagent D and CYP2C19 primer, and triple measurement was performed.
4). Results The results are shown in Table 20.
In the cell recovery group using 300 μL of cell suspension b and the control group using 30 μL of cell suspensions a and b, all were positive for LAMP reaction in triplicate measurement. On the other hand, in the control group using 30 μL of the cell suspension c, all LAMP reactions were negative in triplicate measurement.
5). Discussion When cells are not collected in the cell collection experiment group, it is considered that the LAMP reaction is negative as in the cell suspension c of the control group, but the same results as the cell suspensions a and b are obtained. From this, it was speculated that animal cells were recovered by this method.

According to the present invention, microbial cells or animal cells can be collected inexpensively and easily. In addition, according to the present invention, by collecting and concentrating microbial cells or animal cells, the detection performance of the genetic test kit for microbial cells or animal cells such as pathogenic bacteria can be improved.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

A method for recovering cells, comprising a step of mixing cells and magnetic particles in the presence of alcohol.
The method according to claim 1, wherein the cell is a microorganism or an animal cell.
The method according to claim 1 or 2, wherein the magnetic particles are magnetic particles containing magnetite and / or silica as a surface material.
The alcohol according to any one of claims 1 to 3, wherein the alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol, ethylene glycol, 2,2'-iminodiethanol, polyethylene glycol and mixtures thereof. Method.
The method according to claim 4, wherein the polyethylene glycol has a molecular weight of 20,000 or less.
An inspection method comprising the cell recovery method according to any one of claims 1 to 5.
Cell recovery kit containing magnetic particles and alcohol.
The kit according to claim 7, wherein the cell is a microorganism or an animal cell.
The kit according to claim 7 or 8, wherein the magnetic particles are magnetic particles containing magnetite and / or silica as a surface material.
The alcohol according to any one of claims 7 to 9, wherein the alcohol is selected from the group consisting of methanol, ethanol, 1-propanol, isopropyl alcohol, ethylene glycol, 2,2'-iminodiethanol, polyethylene glycol and mixtures thereof. kit.
The kit according to claim 10, wherein the polyethylene glycol has a molecular weight of 20,000 or less.
A test kit comprising the cell recovery kit according to any one of claims 7 to 11.