JP7220144B2 - Mycoplasma collection method - Google Patents

Description

The present invention relates to a method for collecting mycoplasma that does not use cells.

Mycoplasma refers to bacteria of the genus Mycoplasma, Ureaplasma, Spiroplasma, Acholeplasma, etc., which belong to the network of Molycutis. Mycoplasma is the smallest microorganism capable of self-proliferation and is about one-tenth the size of bacteria (300-1000 nm) (Non-Patent Document 1). Mycoplasma is known as a microorganism that binds to the cell surface of eukaryotic cells and contaminates cultured cells, and mycoplasma contamination poses a serious problem in the production of pharmaceuticals.

Especially in the field of regenerative medicine, cells collected from a subject are cultured, and the cultured cells are either returned to the subject or transferred to a third party's body for treatment. Collateral is essential. The medium used in this case is generally sterilized by autoclave or filter sterilization by applying heat in advance. It is common to sterilize using a filter of However, due to their small size and lack of cell walls, mycoplasma are quite flexible and most cells pass through filters of 450 nm diameter and even 100-200 nm diameter filters. In some cases, filter sterilization cannot completely eliminate mycoplasma (Non-Patent Document 2). Therefore, filter sterilization is not always sufficient to remove mycoplasma, and there is a need for alternative methods of verifying the absence of mycoplasma from a subject after sterilization.

Therefore, the Japanese Pharmacopoeia reference information recommends that cells used in the production of biopharmaceuticals should be tested for mycoplasma by an appropriate method. Methods for denying the existence of mycoplasma include three detection methods: a culture method, a DNA staining method using indicator cells, and a nucleic acid amplification method (Non-Patent Document 3). In particular, nucleic acid amplification methods are listed as detection methods by polymerase chain reaction (PCR). When conducting this mycoplasma denial test, using the fact that mycoplasma adheres to cells and proliferates, from the culture supernatant that may be infected with mycoplasma, Vero cells derived from kidney epithelial cells of African green monkeys As a coprecipitant, a method for collecting mycoplasma has been reported (Non-Patent Document 4). At this time, it has been reported that the collection of mycoplasma is not successful unless Vero cells are added, and it is possible that the collection rate of mycoplasma can be increased by adding something that co-precipitates not only cells but also bacterial bodies. has been reported. In addition, mycoplasma contamination is based on the premise that cell suspensions are used as specimens, but there are cases where valuable cultured cells cannot be used, and methods for detecting mycoplasma liberated from culture supernatants are also being investigated. It is Mycoplasma released into the supernatant during cell culture is usually less than 5%. It has been reported that sensitivity is increased (Non-Patent Document 5).

As a method for collecting cells simply and with high efficiency, it is known to use a protein called MDP1, which binds to sugars on the cell surface, as an aggregation promoter, and it is known that cells can be precipitated without centrifugation. Are known. In addition, a method of adding a solid support has been reported (Patent Document 1).

JP 2010-104250 A

Arihiro Ohara et al., TISSUE CULTURE RESEARCH COMMUNICATIONS, Vol. 26, P159-163 (2007) William B. Whitman, Bergey's Manual of Systematic Bacteriology Second Edition Volume Four, Springer, P575 Japanese Pharmacopoeia 17th Edition Reference Information Yutaka Kikuchi, Health Labor and Welfare Science Research Grant (Research Project for Deregulation and Evaluation of Pharmaceuticals, etc.) 2014 Shared Research Report, Research on Safety Evaluation of Infectious Agents such as Viruses Research on Sterility Test Methods - In Cell and Tissue Processed Products About the Sterility Test Method-, P255-272 Eriko Uchida et al., Bioscience and Industry, Vol. 33, No. 9, P4-14 (2016)

In carrying out the nucleic acid proliferation method as the mycoplasma negative test, the conventionally known mycoplasma collection method using cells as a coprecipitant confirms whether mycoplasma is present in the culture solution to be tested. Therefore, it is necessary to prepare the cells according to the test date of the target culture medium. In addition, it is necessary to confirm in advance that mycoplasma (or other bacteria) are not present in the cells and culture media used. This preparation and confirmation is extremely complicated and prevents convenient testing. It is also known to use Vero cells as a coprecipitant, but in that case, Vero cell-derived DNA, RNA, DNase, RNase, and other proteins are mixed, and after collecting Mycoplasma, It may interfere with the nucleic acid amplification reaction to be performed.
Accordingly, an object of the present invention is to provide a method for reliably collecting mycoplasma in a specimen by simple means without using cells as a coprecipitant.

In view of this situation, the present inventors have made intensive studies to solve the above problems, and found that mycoplasma can be reliably collected as aggregates by adding a protein, preferably a water-insoluble carrier, to the sample. He found this and completed the present invention.

That is, the present invention provides the following [1] to [15].
[1] A method for collecting mycoplasma in a specimen, which comprises adding a protein to the specimen and collecting the resulting aggregates.
[2] The method according to [1] above, wherein the protein is one or more selected from the group consisting of albumin, casein, hydrolyzed casein, milk protein and gelatin.
[3] The method according to [2] above, wherein the albumin is animal serum albumin.
[4] The method according to any one of [1] to [3] above, wherein the amount of protein used is 0.1 to 500 mg per 1 mL of specimen.
[5] Any one of [1] to [4] above, wherein the specimen is a liquid specimen selected from the group consisting of sterilized water, physiological saline, liquid medium, biological sample, culture supernatant, buffer solution and Ringer's solution. The method described in the section.
[6] Mycoplasma is from Acoleplasma leidrowie, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orynis, Mycoplasma oleare, Mycoplasma pneumoniae, Mycoplasma salivarium, Mycoplasma synoviae, Mycoplasma gallisepticum and Spiroplasma citri The method according to any one of the preceding items [1] to [5], which is one or more selected from the group consisting of:
[7] The method according to any one of [1] to [6] above, wherein the collecting means is centrifugation at 3,000 to 30,000 G for 1 to 60 minutes.
[8] A method for measuring mycoplasma, which comprises subjecting mycoplasma collected by the method according to any one of [1] to [7] above to a polymerase chain reaction.
[9] The method according to any one of [1] to [8] above, wherein a water-insoluble carrier is further added to the sample.
[10] The method according to [9] above, wherein the water-insoluble carrier is latex particles, silica particles or colloidal gold particles.
[11] The method according to [10] above, wherein the latex particles are organic polymer latex particles.
[12] The method according to [10] or [11] above, wherein the latex particles have a particle size of 10 to 2,000 nm.
[13] The method according to any one of [9] to [12] above, wherein the amount of the water-insoluble carrier used is 0.0005 to 10 mg per 1 mL of the specimen.
[14] A kit used in the method according to any one of [1] to [8] above, which contains a protein.
[15] A kit used in the method according to any one of [9] to [13] above, further comprising a water-insoluble carrier.

It was confirmed that the method of the present invention has an extremely high collection efficiency of mycoplasma compared to the conventionally known method using cells as a coprecipitant. In addition, the method of the present invention is highly convenient because it is not necessary to prepare cells according to the date of examination, and it is not necessary to confirm that mycoplasma is not present in the cells and medium. Furthermore, the use of a water-insoluble carrier such as latex particles makes it difficult for the aggregates after separation to diffuse and at the same time makes them easy to visually confirm, thereby facilitating removal of the medium after collection of mycoplasma. In addition, since cell-derived DNA, RNA, DNase, RNase, and other proteins are not mixed, nucleic acid amplification reaction operation after collection of mycoplasma is facilitated.

Preferred embodiments of the present invention will be specifically described below. In addition, this invention is not restricted at all by these embodiments.

In the present invention, "collecting mycoplasma" means separating and concentrating mycoplasma contained in a specimen. Collecting the mycoplasma facilitates subsequent mycoplasma detection tests.

In the present specification, the term mycoplasma refers to mycoplasma, which is generally called mycoplasma, and the scientific name refers to a group of microorganisms belonging to the order Mycoplasma tales. Bacteria belonging to the genus Mycoplasma, Ureaplasma, Spiroplasma, Acholeplasma, and the like. In the 17th revised Japanese Pharmacopoeia Reference Information, Acoleplasma laidlawii, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orare ( Mycoplasma orale), Mycoplasma pneumoniae, and Mycoplasma salivarium 7 species 7 strains are used as validation strains, and these strains are 10 cfu / mL or less in the mycoplasma negative test need to confirm. In addition, in European and American pharmacopoeia, Mycoplasma synoviae, Mycoplasma gallisepticum, and Mycoplasma citri are also targeted (European Pharmacopoeia 8th Edition, Theoplasmosis unit National Formulary (USP39-NF34)).

In the present invention, the specimen refers to a sample for testing the presence or absence of mycoplasma infection, and is preferably in liquid form. Examples of specimens include sterilized water, physiological saline, liquid media (for example, those used for culturing cells such as eukaryotic cells), biological samples, culture supernatants, buffers, Ringer's solution, and the like.

Proteins used in the present invention are not particularly limited, but examples include albumins (e.g., bovine serum albumin (BSA), human serum albumin, primate serum albumin, rabbit serum albumin, rodent serum albumin, equine serum albumin, animal albumins such as goat serum albumin, sheep serum albumin, dog serum albumin, guinea pig serum albumin, chicken serum albumin, and porcine serum albumin), casein, hydrolyzed casein, milk protein (for example, product name: Block Ace (DS Pharma Biomedical )), gelatin, and the like. A sterile protein is preferable from the viewpoint of avoiding bacterial contamination when mycoplasma is subjected to PCR after collection. These proteins can be used singly or in combination of two or more.

The amount of protein used is not particularly limited as long as it is an amount that can completely agglutinate mycoplasma. 20 mg.

The water-insoluble carrier used in the present invention is not particularly limited as long as it can agglutinate mycoplasma when used in combination with protein. Polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (Meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylate copolymer, polyvinyl acetate acrylate, etc. latex particles (for example, organic polymer latex particles), latex particles processed for physical adsorption or chemical bonding via carboxyl groups, colored cellulose particles, silica particles, colloidal gold particles, and the like. Among these, latex particles, particularly organic polymer latex particles, are preferred from the viewpoint of cohesion of mycoplasma.

The shape of the water-insoluble carrier is not particularly limited, but a spherical or approximately spherical shape is preferable from the viewpoint of aggregation of mycoplasma.

The particle size of the water-insoluble carrier is not particularly limited, but the average particle size is preferably 10 to 2,000 nm, more preferably 50 to 1,500 nm, still more preferably 50 to 1,500 nm, from the viewpoint of visibility and simplicity of experiment. 100 to 1,000 nm. The particle size of the water-insoluble carrier can be measured, for example, using an electron microscope (TEM) method.

The amount of the water-insoluble carrier used is not particularly limited as long as it is an amount that can completely agglutinate mycoplasma. ~0.1 mg.

The weight ratio of the protein to the water-insoluble carrier is preferably protein:water-insoluble carrier = 1:1 to 50000:1, more preferably 10:1 to 10000:1, still more preferably 10:1 to 10000:1, from the viewpoint of cohesion of mycoplasma. 200:1 to 1000:1.

By adding protein to the specimen, it forms aggregates with mycoplasma in the specimen. At this time, if a water-insoluble carrier is added, aggregates can be formed more efficiently. Mycoplasma can be collected by collecting the obtained aggregates.

Specifically, first, a sample and protein are added to a centrifugation tube. At this time, it is preferable to add a water-insoluble carrier. The specimen is preferably liquid, and water, physiological saline, buffer (for example, Tris-HCl buffer adjusted to a predetermined pH, HEPES buffer, MOPS buffer, HEPPS buffer, TAPS buffer, phosphate buffer (PBS), etc. ), liquid medium (eg, RPMI1640, DMEM, α-MEM, HANKS, etc.), cell suspension supernatant, or culture supernatant after culturing cells. It may be stirred or left for any length of time (eg, 1 second to 60 minutes) to ensure aggregation. Collecting the obtained aggregates can be carried out, for example, by centrifugation. The centrifugal force in centrifugation is not particularly limited, but is, for example, 3,000 to 30,000G, preferably 5,000 to 28,000G, more preferably 14,000 to 25,000G. Centrifugation time is not particularly limited, but is, for example, 10 seconds to 120 minutes, preferably 1 minute to 60 minutes, more preferably 10 minutes to 30 minutes. The temperature during centrifugation is not particularly limited, but is, for example, 4 to 35°C, preferably 7 to 30°C, more preferably 10 to 20°C. By centrifuging, mycoplasma accumulates in the lower part of the centrifugation tube, so the supernatant is removed. If necessary, the steps of centrifuging and removing the supernatant can be repeated two or more times to obtain concentrated mycoplasma as a residue.

In addition, the specimen may optionally be subjected to physical and/or chemical treatments to obtain the aggregates. Physical treatments include heat treatment, ultrasonic irradiation treatment, freezing, melting treatment, and the like. Examples of chemical treatments include chemical reagent treatments, denaturation treatments using digestive enzymes, surfactants, bacteriolytic agents, and the like.

The present invention also relates to a method for measuring mycoplasma by subjecting the mycoplasma-containing residue collected by the above method to polymerase chain reaction (PCR).

In this method, using a mycoplasma-containing residue, DNA is extracted by a known method using a commercially available kit (for example, DNeasy Blood & Tissue Kit from Qiagen (Hilden, Germany)), and a mycoplasma gene detection kit (Myco Finder, Nissui) is used. Seiyaku Co., Ltd. (Tokyo, Japan) can be used to detect mycoplasma. In this detection test, a known PCR method or its modified method (for example, real-time PCR) is used.

The measurement of the present invention can be performed using a kit for mycoplasma collection, which contains a protein, preferably a water-insoluble carrier, for the sample. The protein and water-insoluble carrier contained in the kit are as described above. The kit may optionally be accompanied by a manual explaining the above operations.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1-1, Example 1-2, and Comparative Examples 1-5 Comparative effect test by cells, latex, and protein (1) Reagent and cells (1-1) Water-insoluble carrier Polystyrene latex particles with a particle size of 315 nm ( IMMUTEX (registered trademark, manufactured by JSR Life Sciences Co., Ltd.) concentration 10 w/v%, surface modification: COOH, average particle size measurement method: TEM method) was used.
(1-2) Protein A BSA solution (30 w/v % albumin solution, bovine serum-derived (BSA solution) fatty acid-free, manufactured by Wako Pure Chemical Industries, Ltd.) was used. A calcium ion- and magnesium ion-free phosphate-buffered saline (PBS(-)) was used for dilution.
(1-3) Cell Culture As a control, HEL92.1.7 cells derived from human Caucasian erythroleukemia were used.
(1-4) Specimen As a specimen, PBS(-) containing a specified amount of mycoplasma was used.

(2) Mycoplasma collection method Example 1-1 Using both BSA and latex 50 mL polypropylene tube containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu / mL) PBS (-) (25 mL), 10 w / A v% latex solution (2.5 μL) and a BSA solution (350 μL, 105 mg in terms of BSA) were added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL).

Comparative Example 1 Using conventional cells In a 50 mL polypropylene tube, Mycoplasma salivarium (ATCC23064-TTR) containing (10 cfu/mL) PBS (-) (25 mL), human Caucasian erythroleukemia-derived HEL92.1.7 cells ( 5×10 ⁶ cells/mL, 100 μL) was added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL.
Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). It was previously confirmed that the HEL92.1.7 cells used in the test were not infected with mycoplasma.

Example 1-2 Using BSA PBS (-) (25 mL) containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu/mL), PBS (-) (2.5 μL) and BSA solution (350 μL) in a 50 mL polypropylene tube ) was added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL).

Comparative Example 2 Using Latex In a 50 mL polypropylene tube, Mycoplasma salivarium (ATCC23064-TTR) containing (10 cfu/mL) PBS (-) (25 mL), 10 w/v% latex solution (2.5 μL) and PBS (-) (350 μL) was added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL).

Comparative Example 3 No addition PBS (-) (25 mL) containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu/mL) and PBS (-) (2.85 μL) were added to a 50 mL polypropylene tube, and the mixture was stirred. . The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL).

Comparative Example 4 theoretical value (no concentration step)
Mycoplasma salivarium (ATCC23064-TTR) was added to PBS(−) (200 μL) to 250 cfu and prepared without a concentration step.

(3) Mycoplasma Confirmation Test Test Example Mycoplasma Confirmation Test Mycoplasma concentration obtained in (2) above (200 μL, Example 1-1, Example 1-2 and Comparative Examples 1-5), extraction kit (Qiagen (Hilden, Germany) using the DNeasy Blood & Tissue Kit). The extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20° C.), and the supernatant (25 μL) was used for real-time PCR using Myco Finder manufactured by Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). , the Cq value (number of cycles) was determined (the test was performed twice, and the average value of the Cq values was also determined). This determined the presence or absence of the mycoplasma gene in the sample.

(4) Results (4-1)
Table 1 shows the results.

Since the Cq value when BSA and latex are added (Example 1-1) is about 2 lower than the Cq value when cells are added (Comparative Example 1), Example 1-1 is a comparative example. It was amplified about 2 cycles earlier than 1. Therefore, the method for recovering mycoplasma using BSA and latex can recover four times the amount of PCR template compared to the method using cells.

In addition, when BSA was added (Example 1-2), the Cq value was about 1 lower than when cells were added (Comparative Example 1). It was amplified one cycle earlier than Example 1. Therefore, the method for recovering mycoplasma using BSA can recover twice the amount of PCR template as compared to the method using cells.

Since the Cq value when BSA and latex are added (Example 1-1) is about 6 lower than the Cq value when latex is added (Comparative Example 2), Example 1-1 is a comparative example. It was amplified 6 cycles earlier than 2. Therefore, the method of recovering mycoplasma using BSA and latex can recover 64 times the amount of PCR template compared to the case of using only latex.

Further, when no additional component was added (Comparative Example 3), amplification by PCR was not obtained. Furthermore, since the Cq value of Example 1-1 showed a value close to the theoretical Cq value (Comparative Example 4) without performing the concentration step, the loss due to the concentration step using BSA and latex was less than that of the conventional method. It was extremely small compared to the case of using a certain cell.

In addition, the Cq value of Example 1-2 also showed a close value compared to the theoretical Cq value (Comparative Example 4) where no concentration step was performed and the case of using cells as a conventional method. The loss due to the concentration process using the was less than the conventional method using cells.

Example 2 Examination of Latex Concentration (2-1) Method 50 mL polypropylene tube containing (10 cfu/mL) PBS (-) (25 mL) containing Mycoplasma salivarium (ATCC23064-TTR), 10 w/v% latex solution (as described above) same) and 30 w/v % BSA solution (same as above, 350 μL) were added and the mixture was stirred. At this time, the amount of 10 w/v % latex added was adjusted to 0.5 μL, 1 μL, 2.5 μL, or 25 μL. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). DNA was extracted from the cell concentrate (using QIAamp DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and the supernatant was (25 μL) was used to compare Cq values by real-time PCR method.

(2-2) Results Table 2 shows the results.

From Table 2, it was found that the amount of latex added had almost no effect on the Cq value. Therefore, it is considered that the amount of latex added has little effect on the recovery amount of the PCR template.

Example 3 Examination of water-insoluble carriers (silica particles, gold colloid particles)
(3-1) Method 50 mL polypropylene tube containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu / mL) PBS (-) (25 mL), water-insoluble carrier and 30 w / v% BSA solution (same as above, 350 μL) was added and the mixture was stirred. At this time, the water-insoluble carrier is a 10 w/v% latex solution (same as above, 2.5 μL), 50 mg/mL silica particles (Sicastar 300 nm (manufactured by Micromod), average particle diameter 300 nm, surface modification: COOH , Average particle size measurement method: dynamic light scattering method, 5.0 μL), 0.124 mg / mL colloidal gold particles (300 nm standard gold nanoparticles (manufactured by Cyto Diagnostics), average particle size 300 nm, surface treatment : none, average particle size measurement method: dynamic light scattering method, 2 mL) was used. Since the amount of colloidal gold particles added was large, the sample amount was set to 23 mL. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and Cq values were compared by the real-time PCR method using the supernatant (25 μL).
Also, HEL92.1.7 cells (5×10 ⁶ cells/mL, 100 μL) were added instead of 10 w/v% latex solution (2.5 μL) and 30 w/v% BSA solution (350 μL), respectively. Subject measured.

(3-2) Results Table 3 shows the results.

The content of solid components is as follows (calculated value).
Latex particles: 0.25 mg
Silica particles: 0.25 mg
Gold colloid particles: 0.248 mg
From Table 3, it was found that mycoplasma can be detected using silica particles and colloidal gold particles as well as latex particles.

Example 4 Examination of BSA concentration (4-1) Method 50 mL polypropylene tube containing Mycoplasma salivarium (ATCC23064-TTR) (8 cfu / mL) PBS (-) (25 mL), 10 w / v% latex solution ( 2.5 μL, same as above) and 30 w/v % BSA solution (same as above) were added and the mixture was stirred. At this time, the amount of BSA solution added was adjusted to 0 μL (0 g/25 mL), 175 μL (0.05 g/25 mL), 350 μL (0.1 g/25 mL), or 1,500 μL (0.5 g/25 mL). bottom. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using DNeasy Blood & Tissue Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and the supernatant ( 25 μL) were used to compare Cq values by real-time PCR.

(4-2) Results Table 4 shows the results.

From Table 4, compared with the case of using only latex, at a BSA concentration of 0.05 g/25 mL or more, the pellet after centrifugation was firm and there was no significant difference in the Cq value.

Example 5 Investigation of BSA low concentration (5-1) Method A 50 mL polypropylene tube containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu / mL) PBS (-) (25 mL), 10 w / v% latex solution (above 2.5 μL) and 30 w/v % BSA solution (same as above) were added and the mixture was stirred. At this time, the amount of BSA solution added was adjusted to 17.5 μL (0.005 g/25 mL), 35 μL (0.01 g/25 mL), or 350 μL (0.1 g/25 mL). The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and Cq values were compared by the real-time PCR method using the supernatant (25 μL).

(5-2) Results Table 5 shows the results.

From Table 5, compared to the case of using only latex, when BSA was added, variations in the Cq value were observed at concentrations of 0.01 g/25 mL and 0.005 g/mL, and the pellet after centrifugation Although looseness was observed, it was determined that mycoplasma collection was possible.

Example 6 Examination of protein (Block Ace)
(6-1) Method In a 50 mL polypropylene tube, 25 mL of PBS (-) containing Mycoplasma salivarium (ATCC23064-TTR) (8 cfu / mL), 10 w / v% latex solution (same as above, 2.5 μL) and milk The protein Block Ace (manufactured by DS Biomedical) was added and the mixture was stirred. At this time, the amount of Block Ace added was adjusted to 0 g/mL, 1 g/25 mL, or 0.1 g/25 mL. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using DNeasy Blood & Tissue Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and the supernatant ( 25 μL) were used to compare Cq values by real-time PCR.

(6-2) Results Table 6 shows the results.

From Table 6, the Cq value was about 2 lower when Block Ace was added than when only latex was used, regardless of the Block Ace concentration.

Example 7 Examination of protein (gelatin)
(7-1) Method 50 mL polypropylene tube containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu / mL) PBS (-) (25 mL), 10 w / v% latex solution (same as above, 2.5 μL) and A 10% gelatin (from cold water fish skin, 40-50% in H ₂ O, Sigma-Aldrich) solution was added and the mixture was stirred.At this time, the 10% gelatin solution was added. The volumes were adjusted to 525 μL (0.05 g/25 mL), 1,050 μL (0.1 g/25 mL), 5,250 μL (0.5 g/25 mL).The stirred sample was then heated at 20,000 G. After centrifugation (10° C.) for 30 minutes, 24.5 mL of the supernatant was removed, and the pellet was resuspended.The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Further, this solution was centrifuged at 20,000 G (20° C.) for 10 minutes, and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). Extraction (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), centrifugation of the extracted DNA solution at 20,000 G for 15 minutes (20° C.), and real-time PCR using the supernatant (25 μL). Cq values were compared by the method.

(7-2) Results Table 7 shows the results.

From Table 7, it was found that mycoplasma can be harvested in a concentration-dependent manner when gelatin is used as the protein instead of BSA. Equivalent Cq values were obtained with the addition of 0.5 g/25 mL gelatin compared to the use of 0.1 g/mL BSA alone.

Example 8 Study using polybacterial species (8-1) Method Acholeplasma laidlowi (ATCC23206-TTR), Mycoplasma arginini (ATCC23838-TTR), Mycoplasma fermentans (ATCC19989-TTR) in a 50 mL polypropylene tube , Mycoplasma orynis (ATCC17981-TTR), Mycoplasma olere (ATCC23714-TTR), Mycoplasma pneumoniae (ATCC15531-TTR) each containing (10cfu/mL) PBS (-) (25mL), 10w/v% latex solution ( 2.5 μL, same as above) and 30 w/v % BSA solution (350 μL, same as above) were added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Further, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), 1,200 μL of the supernatant was removed, and a bacterial cell concentrate (200 μL) was obtained. From the cell concentrate, DNA was extracted (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and Cq values were compared by the real-time PCR method using the supernatant (25 μL). Also, HEL92.1.7 cells (5×10 ⁶ cells/mL, 100 μL) were added instead of 10 w/v% latex solution (2.5 μL) and 30 w/v% BSA solution (350 μL), respectively. Subject measured.

(8-2) Results Table 8 shows the results.

In the Japanese Pharmacopoeia, 7 bacterial species are problematic as described above, but from Table 8, by using the method of the present invention, not only Mycoplasma salivarium shown in Example 1, but also Acholeplasma laidlowi, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orynis, Mycoplasma orale, and Mycoplasma pneumoniae were also found to be detectable.

Example 9 Examination using AIM-V medium or KBM medium as a specimen (9-1) Method AIM-V medium (AIM-V) containing Mycoplasma salivarium (ATCC23064-TTR) (10 cfu/mL) in a 50 mL polypropylene tube Medium CTS ^™ , Life Technologies, 25 mL) or KBM medium (KBM550, Kohjin Bio, 25 mL), 10 w/v% latex solution (same as above, 2.5 μL) and 30 w/v% BSA solution (above 350 μL) was added and the mixture was stirred. The stirred sample was then centrifuged at 20,000 G for 30 minutes (10° C.) and the pellet was resuspended after removing 24.5 mL of supernatant. The resuspension was transferred to a 1.5 mL polypropylene tube, and PBS(-) was added to bring the total volume to 1,400 μL. Furthermore, this liquid was centrifuged at 20,000 G for 10 minutes (20° C.), and 1,200 μL of the supernatant was removed to obtain a bacterial cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and Cq values were compared by the real-time PCR method using the supernatant (25 μL). In addition, HEL92.1.7 cells (5 × 10 ⁶ Cells/mL, 100 µL) were added instead of 10 w/v% latex solution (2.5 µL) and 30 w/v% BSA solution (350 µL). Subject measured.

(9-2) Results Table 9 shows the results.

From Table 9, it was confirmed that mycoplasma can be collected even if the specimen is AIM-V medium or KBM medium.

Example 10 Small Volume Study (10-1) Method A 5 mL polypropylene tube containing (10 cfu/mL) specimen (1 mL) containing Mycoplasma pneumoniae (ATCC15531-TTR), 10 w/v% latex solution (same as above, 2 .5 μL) and 30 w/% BSA solution (same as above, 14 μL) were added and the mixture was stirred. At this time, AIM-V medium (same as above), KBM medium (same as above), Ringer's solution (lact Ringer's solution "Fuso", manufactured by Fuso Pharmaceutical Co., Ltd.), physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Co., Ltd.) were used as samples. company) and PBS (-) were used. Next, the stirred sample was centrifuged at 20,000 G for 30 minutes (10° C.), and 816.5 μL of the supernatant was removed to obtain a cell concentrate (200 μL). From the cell concentrate, DNA was extracted (using QIAamp UCP DNA Micro Kit from Qiagen (Hilden, Germany)), the extracted DNA solution was centrifuged at 20,000 G for 15 minutes (20°C), and Cq values were compared by real-time PCR method using the supernatant (25 μL) (average of 6 replicates).

(10-2) Results Table 10 shows the results.

From Table 10, AIM-V medium, KBM medium, Ringer's solution, saline, and PBS (-) confirmed that mycoplasma can be collected even from a 1 mL sample containing a small amount of mycoplasma. was done.

Claims (13)

A liquid sample selected from the group consisting of sterilized water, physiological saline, liquid medium, biological sample, culture supernatant, buffer solution and Ringer's solution, albumin, casein, hydrolyzed casein, milk protein and gelatin selected from the group 0.1 to 500 mg of one or more proteins are added to 1 mL of the specimen, and the resulting aggregates are collected by centrifugation at 3,000 to 30,000 G for 1 to 60 minutes . A method for collecting mycoplasma in a sample. 2. The method of claim 1, wherein the albumin is animal serum albumin. 3. The method according to claim 1 or 2, wherein the specimen is a liquid specimen selected from the group consisting of physiological saline, liquid medium, biological sample, culture supernatant, buffer solution and Ringer's solution. Mycoplasma is from the group consisting of Acoleplasma laidlowi, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orynis, Mycoplasma orare, Mycoplasma pneumoniae, Mycoplasma salivarium, Mycoplasma synoviae, Mycoplasma gallisepticum and Spiroplasma citri The method according to any one of claims 1 to 3, which is one or more selected. The method according to any one of claims 1 to 4, wherein the collecting means is centrifugation at 5,000 to 28,000 G for 1 minute to 60 minutes. A method for measuring mycoplasma, which comprises subjecting mycoplasma collected by the method according to any one of claims 1 to 5 to a polymerase chain reaction. The method according to any one of claims 1 to 6, wherein a water-insoluble carrier is further added to the sample. 8. The method according to claim 7, wherein the water-insoluble carrier is latex particles, silica particles or colloidal gold particles. 9. The method of claim 8, wherein the latex particles are organic polymeric latex particles. The method according to claim 8 or 9, wherein the latex particles have a particle size of 10 to 2,000 nm. The method according to any one of claims 7 to 10, wherein the amount of water-insoluble carrier used is 0.0005 to 10 mg per 1 mL of the specimen. A kit for use in the method according to any one of claims 1 to 7, comprising one or more proteins selected from the group consisting of albumin, casein, hydrolyzed casein, milk protein and gelatin. 13. The kit of claim 12, further comprising a water-insoluble carrier.