JP4987469B2 - Assay to detect mycoplasma by measuring acetate kinase or carbamate kinase activity - Google Patents

Description

  The present invention relates to assay methods and materials for detecting Mollicutes family members that contaminate test samples, such as samples from cell cultures.

  Taxonomically, the lack of cell walls has been used to separate Mollicutes from other bacteria in steel called Mollicutes (Razin et al. 1998). The steel constituents are summarized in Table 1 below.

  In the context of the present application, the term “mycoplasma” is intended to include all constituents of Mollicutes steel, not just mycoplasma. In fact, “mycoplasma” is a general term for all Mollicutes in the field.

  Mycoplasma is widespread in nature as a parasite of humans, mammals, reptiles, fish, arthropods and plants. Mycoplasma is the smallest and simplest prokaryote. Mycoplasma lacks a tight cell wall and is unable to synthesize peptidoglycan; therefore, mycoplasma is not sensitive to antibiotics such as penicillin and its analogs. Mycoplasma was generated by degenerative evolution from two species of Gram-positive bacteria with low guanine and cytosine content of DNA, namely Lactobacillus, Bacillus, Streptococcus and Clostridium. Mollicutes have lost a significant portion of their genetic information during the course of evolution. It is this limited coding ability that determines the need for a parasitic life cycle. Most species are facultative anaerobes, but some are absolute, and therefore their metabolism is similar to anaerobic bacteria.

Over 180 Mollicute species have been identified, in which 20 different mycoplasma and Acholeplasma species from human, bovine and porcine have been isolated from cell cultures. There are six species that account for 95% of all mycoplasma infections; these are M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii. The main cause of infection is cross-contamination from other cell lines brought into the laboratory. In addition, undesirable sources of exogenous mycoplasma can be found in tissue culture reagents such as serum products. Mycoplasmas, unlike bacteria, are less likely to produce increased turbidity or obvious cell damage. Viable mycoplasma can be recovered from the working surface 7 days after inoculation, and the mycoplasma can also pass through a bacteria retaining filter. In its maximum individual phase, mycoplasma can be present as much as 10 ⁸ mycoplasma / ml supernatant at a 5: 1 ratio to the host cells. When present, the mycoplasma “grows” to a detectable concentration in the culture medium, which is then further adsorbed onto the cell surface. This is an issue as to whether mycoplasma enters and survives mammalian cells in culture.

  Mycoplasma can alter almost each property of in vitro cultures. Mycoplasma depletes culture nutrients, especially arginine. Infected eukaryotic cells exhibit abnormal growth, metabolism and morphological changes. Several biological properties have been implicated as toxicity determinants; these include the secretion or introduction of mycoplasma enzymes such as phospholipases, ATPases, hemolysins, proteases and nucleases into the host cell environment.

  The main problem with mycoplasmas is that their contamination is often invisible and, unlike bacterial detection, cannot be easily seen with the naked eye. Mycoplasma's resistance to antibiotics and the ability to pass normal bacterial sterilization filters means that mycoplasma can escape the typical precautions of cell culture techniques. As a result of the negative effects that these contaminations are still undetected, it has become apparent that continuous screening is essential for any cell culture laboratory.

  There are several studies (Rottem and Barile 1993, McGarrity and Kotani 1985) that have shown that at least 10% to 15% of cells in culture can be contaminated by mycoplasms. Most cell biologists understand the need to conduct routine tests on mycoplas, however, due to the cost and inaccuracy of currently available tests, this has still been realized so far. Not remain ideal.

  The only accurate method available for detecting viable mycoplasma is culturing microorganisms. However, the difficulties associated with their in vitro culture have proven problematic due to the complex media required for their culture (Razin et al., 1998). Culture has also been considered the most sensitive method. This is because it is said that a single living microorganism can be detected. However, the results take 2-3 weeks, depending on the skilled person and very specific culture requirements. Such time is a result of the need to culture the cells to a sufficient number that the cells form colonies, and this can therefore be identified using Deans staining. Mycoplasma can be cultivated in a broth culture on agar, and most mycoplasma produce microscopic colonies with the characteristic “fried egg” appearance and grow in an embedded state in the agar. However, some colonies may not grow completely embedded in the agar. There are several strains that cannot be easily grown using standard agar or broth culture media. These strains require cell-assisted culture in order to isolate and identify them. The latter approach helps to identify and detect mycoplasma species that adsorb to the host cell surface (Rottem and Barile 1993). However, due to the complex nature of the culture procedure, these tests are most commonly performed by mycoplasma contract laboratories.

  One simple means of detecting mycoplasma in a sample is an assay for DNA using a fluorescent dye. One of the most commonly used is 4 ′, 6-diamine-2-phenylindole dihydrochloride (DAPI), but Hoechst staining is considered to be the selection method. Cell culture samples are taken, fixed and stained with Hoechst 33258 (bisbenzamide) and examined under UV epifluorescence (Battaglia et al., 1994, Raab 1999). In the presence of mycoplasma associated with cells, the cell nucleus thus appears surrounded by fluorescent structures in the cytoplasm. Negative cells are represented by simple nuclear staining of cellular DNA. Accurate interpretation of results from DNA staining requires an experienced eye, which further requires specialized equipment, ie a fluorescence microscope.

  Mycoplasma detection by PCR is a test commonly used by external service laboratories and is also performed in laboratories with appropriate equipment. Primers used in the Mycoplasma PCR kit anneal to conserved regions of the Mycoplasma genome, allowing detection of several species (Raab 1999). Most commercially available PCR kits require the amplified product to be analyzed by agarose gel electrophoresis, and there is a resulting band pattern that determines contaminating species. However, the visualization of the band pattern is subjective.

  The Mycoplasma PCR ELISA (Raab 1999) from Roche relies on different systems and cannot distinguish between species. The kit contains digoxigenin-dUTP and the PCR product is captured on the surface of the wells in a microtiter plate coated with anti-digoxigenin-peroxide complex. The product colored with tetramethylbenzidine (TMB) is made visible using a standard ELISA plate reader.

  Life Technologies has developed a MYCOTECT ™ kit (Verhoef et al., 1983) based on the activity of adenosine phosphorylase, which is found in very small amounts in mammalian cells (sometimes not seen at all). This enzyme converts 6-methylpurine deoxyribose (6-MPDR) into two toxic products (6-methylpurine and 6-methylpurine ribose). This assay requires the addition of contaminating cell lines to indicator cell lines grown in 24-well tissue culture plates. Since these toxic substances are produced when they are positive for mycoplasma, 6-MPDR substrate is added, and after further growth for 3 to 4 days, crystal violet staining solution is added to test the viability of the indicator cells. Although it has been reported to detect one mycoplasma cell per 200,000 target cells, when media conditions are adjusted to facilitate mycoplasma growth (Whitaker et al., 1987), the main disadvantage of this system is It's time consuming and time consuming.

  Mycoplasma antigens can be detected using an immunoassay that utilizes antibodies raised against the mycoplasma antigen. For example, the detection of M. pneumoniae in clinical samples (Daxboeck et al., 2003), the use of different antibodies allows species identification. There are several commercially available kits, for example, IDEXX Laboratories (USA) is an enzyme-linked immunosorbent assay (ELISA) for detecting several mycoplasmas associated with animal health. Supply.

  Most of the known assays take at least 24 hours to complete and require expensive equipment and a considerable amount of expertise. In addition, these are staining specific assays. There is no general assay, ie, one that has the ability to detect mycoplasma species roughly.

  GB 2357336B describes an assay that can be used to detect mycoplasma in cell cultures. This assay is based on the observation that mycoplasma overproduces the enzyme ATPase in large amounts. Mycoplasma ATPase activity converts enough cellular ATP or exogenously added ATP to ADP, making ADP detectable. This assay is therefore based on the detection of ADP, which converts the ADP to ATP, a reagent-containing enzyme (pyruvate kinase and phosphoenolpyruvate; adenylate kinase; glycerol kinase, myokinase combination; or creatine kinase And creatine phosphate) is added to the sample and a bioluminescent reaction is used to detect ATP.

The disclosure of British Patent No. 2357336 is incorporated herein, including possible amendment purposes.
British Patent No. 2357336B British Patent No. 2357336 de Wet et al., 1987, Masuda et al., 1989, Wood et al., 1989, European Patent Application No. 0353464 White et al., 1996, WO 01/31028 and WO 00/24878. www.unc.edu/depts/tcf/mycoplasma.htm

  The present invention seeks to provide other means for detecting mycoplasma in samples, such as samples from cell cultures.

According to a first aspect, the present invention is a method for detecting the presence of contaminating mycoplasma in a test sample comprising:
(i) preparing a test sample;
(ii) detecting and / or measuring the activity (B) of acetate kinase and / or carbamate kinase in the test sample, said activity indicating the presence of contaminating mycoplasma; and
(iii) providing a method comprising verifying that the test sample is contaminated with mycoplasma based on the detection and / or measurement of said activity in step (ii).

Preferably, the method comprises the following steps performed after step (ii) but before step (iii):
(iia) obtaining information (A) of acetate kinase and / or carbamate kinase activity detected and / or measured in a corresponding control sample; and
(iib) comparing the activity (B) detected and / or measured in the test sample with the activity (A) in the control sample;
If the activity (B) detected and / or measured in the test sample during step (ii) is greater than the activity (A) in the control sample during step (iia), ie the ratio B / A is greater than 1 If so, the method further includes the step (iii) of confirming that the test sample is contaminated with mycoplasma.

  In a second aspect, the present invention provides for the detection and / or measurement of acetate kinase and / or carbamate kinase activity (B) in the test sample during step (ii) and / or the corresponding during step (iia) Obtaining information on acetate kinase and / or carbamate kinase activity (A) in the control sample is one or more substrates and / or the following reactions:

  A method comprising detecting and / or measuring the appearance and / or disappearance of one or more products of the present invention.

  Preferably, the detecting and / or measuring step comprises detecting and / or measuring ATP. Even more preferably, ATP is detected and / or measured by a luminescent reaction, in particular a bioluminescent reaction.

  Several luminescent systems are known, some bacteria, protozoa, coelenterate, mollusks, fish, millipede, flies, fungi, helminths, crustaceans, and beetles, especially Photinus, Photuris, and Luciola It has been isolated from many luminescent organisms, including the fireflies of the genus and the click beetle of the genus pyrophorus. In many of these organisms, enzyme-catalyzed redox occurs in which free energy changes are utilized to excite molecules to a high energy state. Thus, visible light is emitted when the excited molecule naturally returns to the ground state. This emitted light is called “bioluminescence”.

Beetle luciferases, especially luciferases from the firefly species Photinus pyralis, have been an example for understanding bioluminescence since early work. P.pyralis luciferase is probably an enzyme with 550 amino acids and a molecular weight of about 60,000 daltons, probably without prosthetic groups or tightly bound metal ions, which has been in crystalline form for many years. Available in the field. By studying the molecular elements in the mechanism of firefly luciferase in generating bioluminescence, the substrate for this enzyme is firefly luciferin, heterocyclic organic acids, D-(-)-2- (6'-hydroxy-2'- It has been shown to be benzothiazolyl) Δ ² -thiazoline-4-carboxylic acid (hereinafter referred to herein as “luciferin” unless otherwise indicated).

  The following bioluminescent reaction can be used to detect ATP.

  The intensity of the emitted light is directly related to the ATP concentration and is measured using a luminometer.

Luciferase has been used as a means of assaying the concentration of ATP, and as little as ^10-16 moles of ATP can be detected using a high quality preparation of this enzyme. The luciferase-luciferin reaction is very specific for ATP. For example, deoxy-ATP produces less than 2% of the light produced by ATP and other nucleoside triphosphates produce less than 0.1%.

  Crystalline luciferase can be isolated directly from the beetle luminescent organs. (In particular, P.pyralis luciferase, P.plagiophthalamus luciferase isozymes, L.cruciata luciferase and L.lateralis luciferase (de Wet et al., 1987, Masuda et al., 1989 CDNAs encoding luciferases of several beetle species are available (including Wood et al., 1989, European Patent Publication No. 0353464). Furthermore, luciferase-encoding cDNAs of any other beetle species that produce luciferase are readily available by those skilled in the art using known techniques (de Wet et al., 1986, Wood et al., 1989). By obtaining a cDNA encoding beetle luciferase, it is obtained in a very pure form by isolating it from bacteria in culture (e.g. E. coli), yeast, mammalian cells, etc. transformed to express the cDNA. It is very easy to prepare a large amount of luciferase with.

  In addition, the availability of cDNA encoding beetle luciferase and the ability to rapidly screen cDNA encoding an enzyme that catalyzes the luciferase-luciferin reaction (see De Wet et al., 1986, supra, Wood et al., Supra). Can also be used to prepare and obtain large quantities of pure mutated luciferase that retains the activity of catalyzing the production of bioluminescence by the luciferase-luciferin reaction. Such a mutant luciferase will have an amino acid sequence that differs from the naturally occurring beetle luciferase sequence at one or more positions (White et al., 1996, WO 01/31028 and WO No. 00/24878). In the present disclosure, the term “luciferase” refers not only to the luciferase originally present in beetles, but also mutants that retain the activity of imparting bioluminescence by catalyzing the luciferase-luciferin reaction of such natural luciferases. Including.

  In the method of the invention, most preferably after step (i), but before step (ii), the sample is treated to dissolve any mycoplasma, thereby releasing its cell contents into the sample. . One skilled in the art will appreciate that lysis can be performed by a variety of methods, including the application of chemicals such as surfactants, and mechanical methods such as sonication.

  It is advantageous to perform the lysis by treating the sample with a detergent or by other lysis methods that can dissolve the mycoplasma cell membrane but do not affect the cell walls of any bacteria that may be present. It is. A typical surfactant treatment involves the use of a low concentration (eg, 0.25% v / v) surfactant such as Triton X100.

  The preferred lysis method is sufficient to dissolve the mycoplasma membrane, but this method does not appear to be effective against bacterial cells. In tests comparing eukaryotic cell lysis to bacterial lysis, non-ionic surfactants (mainly polyethoxyethers) can be used to lyse somatic cells without affecting microbial cells. Have been recognized (Schramm and Weyens-van Witzenberg 1989, Stanley 1989). What makes bacteria insensitive to detergent lysis is the presence of a tight cell wall, requiring a more stringent lysis procedure to lyse the bacterial cells. Bacteria often require exposure to enzymes such as isozymes to disrupt the cell wall for efficient lysis and overall protein release (Pellegrini et al., 1992). The most preferred surfactant dissolution conditions for mycoplasma are given hereinafter.

  However, bacterial contamination produces an increase in turbidity, and the bacteria can also be seen with the naked eye when viewing the cell culture under a phase contrast microscope. These bacterial cultures can be detected very easily and can be discarded immediately.

  Unlike bacteria, mycoplasma passes through a 0.45 μM filter used for filter sterilization (Baseman and Tully 1997), and by adding a filtration step, bacterial and mycoplasma contamination can be distinguished.

  Thus, in a preferred embodiment of the invention, the test sample is passed through a bacterial filter in step (i). Of course, one skilled in the art would not selectively dissolve the mycoplasma when the test sample is treated to remove bacteria, for example by passing the test sample through a bacteria-retaining filter, i.e. dissolve the bacteria. It will be understood that there is no need.

  In a preferred embodiment, ADP is added to the test sample prior to detection and / or measurement step (ii). However, the assay can also utilize endogenous ADP.

  In a preferred embodiment, a mycoplasma substrate reagent is added to the test sample prior to detection and / or measurement step (ii), the mycoplasma substrate reagent being acetyl phosphate or precursor thereof, and / or carbamoyl phosphate or precursor thereof. Including the body.

  By “its precursor” we shall include one or more compounds from which acetyl phosphate and / or carbamoyl phosphate can be generated. A typical reaction is outlined below:

  Thus, instead of adding acetyl phosphate to the mycoplasma substrate reagent, it would be possible to include a precursor such as acetyl CoA.

  Similarly, instead of carbamoyl phosphate, precursors such as citrulline and ammonia could be added to the mycoplasma substrate reagent.

  In the method of the present invention, it is most preferred that acetyl phosphate and carbamoyl phosphate, and / or their precursors are added to the sample prior to step (ii). This allows a general assay for mycoplasma contamination. This is because mycoplasmas utilize one or both substrates by their acetate kinase and / or carbamate kinase enzymes.

  Alternatively, more specific assays can be generated by simply using the aforementioned substrate or one of its precursors. Such an assay would be specific for mycoplasma using only one of the enzyme acetate kinase or carbamate kinase. Table 2 below lists examples of some members of the Mollicutes family (parasitic mammalian hosts) that utilize acetate kinase preferentially, carbamate kinase preferentially, or both. In addition to those listed below, there are infectious mycoplasmas in several reptiles, insects and plants whose biochemical investigation confirmed the use of these same pathways (Kirchoff et al., 1997, Forsyth Et al., 1996, Taylor et al., 1996 and Tully et al., 1994).

  Most preferably, in all methods of all embodiments of the invention, the `` corresponding control sample '' is a mycoplasma lysis treatment, and / or the addition of a mycoplasma substrate, and / or a time interval (e.g., greater than about 30 minutes). The previous test sample. In this preferred embodiment, both activity measurements are made on the same sample, the test sample. A first activity measurement (A) is obtained before or at the same time as the mycoplasma lysis step and then after adding the mycoplasma substrate and / or after a certain time interval (e.g. over about 30 minutes) A second activity measurement (B) is obtained. If the B / A value is greater than 1, confirm that the test sample is contaminated with mycoplasma.

  One skilled in the art will appreciate that a “corresponding control sample” may be a given negative control sample, but this is less preferred.

  In one embodiment, the control sample has been shown to be free of mycoplasma contamination. Suitable methods for doing this include PCR testing, DNA fluorescent staining, or culture methods as described herein. Thus, in one embodiment, a “corresponding control sample” means that we include approximately the same material as that contained in the test sample, but unlike the test sample, it has been shown to be free of mycoplasma contamination. Means. One skilled in the art will appreciate that the mycoplasma uncontaminated state can be indicated by a variety of known methods. Several suitable methods are reviewed by Rottem and Barile 1993, while an overview of test kits and services is given in Raab et al 1999.

  The test sample and / or control sample may be a cell sample, such as a cell culture sample, particularly a culture of mammalian cells. Some examples are listed in Table 3 below.

  In addition, it would be possible to test mammalian primary cell types, as well as all such cells maintained by tissue banks such as ATCC and ECACC.

  Using the assays of the present invention, in cultures of adherent cells (e.g., HepG2, A549, CHO and COS cells), and cells cultured in suspension (e.g., Jurkats, U937, K562 and HL-60 cells) It is noteworthy that mycoplasma contamination can be detected.

  The sample to be tested is preferably derived from a cell culture supernatant which has been previously centrifuged to remove cellular material. However, the assay can also be performed in the presence of cells or cell debris.

  Cell-free samples can also be tested using the method of the present invention. For example, the methods of the present invention include samples of cell-free reagents, such as tissue culture media, and samples typically containing serum (eg, fetal bovine serum), animal-derived materials such as trypsin, and other supplements. Very useful for testing. Examples of some commonly used media and supplements that can be tested in this way are shown in Table 4.

A third aspect of the invention is a method for detecting the presence of mycoplasma in a test sample, comprising the following steps:
(i) preparing a test sample;
(ii) detecting and / or measuring ATP in a test sample using a bioluminescent reaction without adding an exogenous reagent (e.g., a substrate for kinase activity) to convert ADP to ATP; Or obtaining a light output measurement (A),
(iii) obtaining a measurement (B) of ATP and / or light output from the corresponding control sample;
(iv) comparing the ratio B / A of the measured values of ATP and / or light output; and
(v) If the ratio B / A is greater than 1, a method is provided comprising the step of confirming that the test sample is contaminated with mycoplasma.

  As described with respect to the previous embodiments of the present invention, it is most preferred that the mycoplasma lysis treatment and / or the addition of mycoplasma substrate and / or a time interval be performed prior to step (ii).

  As described with respect to previous embodiments of the invention, a `` corresponding control sample '' is subjected to a mycoplasma lysis treatment and / or the addition of a mycoplasma substrate and / or standing at certain time intervals (e.g., greater than about 30 minutes). Most preferably, it is an untested test sample.

  In other words, both measurements are taken from the test sample. Thus, in a preferred embodiment, control ATP and / or light output measurements are obtained after addition of the mycoplasma detection reagent to the sample, including detergent and luciferase / luciferin and AMP, to obtain test ATP and / or light output. Is obtained after addition of a substrate for kinase activity (or a precursor thereof).

  A fourth aspect of the present invention is an in vitro method for treating a cell culture to remove mycoplasma contamination, wherein the mycoplasma contaminated cell culture is treated with an agent to remove and / or remove mycoplasma. Or using the method of the invention to later test the sample from the culture for mycoplasma contamination, if necessary, this process once until no mycoplasma contamination is detected in the sample. Or provide a method comprising repeating multiple times.

  The most common antibiotics used in cell culture are ineffective against mycoplasma. There are several substances that exhibit inhibitory activity, including gentamicin sulfate, kanamycin sulfate and tyrosine tartaric acid (www.unc.edu/depts/tcf/mycoplasma.htm). Several commercially available therapeutic products exist, including mycoplasma scavenger (ICN-Flow), quinolone antibiotic derivatives, and non-antibiotic therapeutic agent Mynox® from Minerva Biolabs (Berlin, Germany) To do. The US company Invivogen supplies Plasmocin ™, which has two bacterial elements, one that acts on protein synthesis and the other that inhibits DNA replication. The antibiotics tetracycline and ciprofloxacin have been reported to have a success rate of less than 80-85% (www.unc.edu/depts/tcf/mycoplasma.htm). Therefore, once contamination is established, it is very difficult to completely eradicate mycoplasma from the culture.

  Most of the effective antibiotics are quinolone derivatives, and the effectiveness of different antibiotics varies according to the mycoplasma species being tested. Duffy et al. 2000 examined the viability of M. pneumoniae, M. hominis, M. fermentans, M. genitalium and U. Compared with antibiotics. These results showed various variability between species, however, gemifloxacin worked better than tetracycline. Because of the tetM gene, there are several species that are resistant to tetracycline. This happens frequently and is complicated by species variability variations that depend on the mycoplasma source. For example, mycoplasmas exposed to antibiotics in eukaryotic cell cultures exhibit a different profile than the same species isolated from human or animal sources (Taylor-Robinson and Bebear 1997). The reported success of anti-mycoplasma treatment appears to vary greatly, but a recent study by Uphoff et al., 2002, found that 96% of leukemia-lymphoma cell lines depend on at least one of the therapeutic agents tested. Reports that mycoplasma is gone.

  Several examples embodying various aspects of the present invention will now be described with reference to the accompanying drawings.

Assay Method of the Invention The principle of the preferred assay method is to provide an appropriate substrate for the mycoplasma enzyme. In the presence of mycoplasma contamination, there is an ADP to ATP conversion which can then be measured, preferably by the luciferase-luciferin reaction.

  Add mycoplasma detection reagent, and after about 5 minutes, obtain a first light output reading (A), add mycoplasma substrate (MS), allow any enzyme activity to proceed for about 10 minutes, at this point the second Get the light reading (B).

  In the presence of mycoplasma contamination, the second reading (B) is therefore large compared to the first reading (A), giving a ratio B / A greater than 1. If the culture is negative (not contaminated by mycoplasma), the ratio B / A is therefore 1 or most often less than 1 due to the decay of the luminescent signal normally seen over time. It will be. FIG. 1 shows the kinetics of this reaction. Typically, the ratio B / A found for mycoplasma contamination is much greater than 1, for example, FIG.

  A preferred assay kit of the present invention comprises a mycoplasma detection reagent (MDR), a mycoplasma assay buffer (MAB) to restore the MDR, and a mycoplasma substrate (MS). MDR and MS are preferably provided as lyophilized preparations.

  All mycoplasmas produce ATP through the acetate kinase pathway or the carbamate kinase pathway. The mycoplasma substrate of the present invention includes one or both of these enzymatic reactions. ADP is necessary for both enzymes and is preferably supplied in excess in the mycoplasma detection reagent of the present invention to promote the occurrence of ATP formation.

  MDR is added to a sample of culture supernatant that has been previously centrifuged to remove cellular material, but the assay can be performed in the presence of cells. Alternatively or additionally, the test sample can be passed through a bacterial filter.

  MDR includes luciferase substrates, luciferin and other cofactors, and AMP and ADP. Mycoplasma substrate (MS) contains carbamoyl phosphate and / or acetyl phosphate or precursors thereof necessary to detect carbamate and / or acetate kinase activity.

  A preferred sample volume is 100 μl, to which 100 μl of reduced MDR is added. After about 5 minutes, a first emission reading (A) is obtained, which gives a basic reading, which determines a further ratio calculation B / A.

  The assay of the present invention has been used to investigate contamination by Acholeplasma laidlawii, M. hyorhinis, M. fermentans, M. orale and M. genitalium and detect some unknown mycoplasma contamination.

  The inventor compared their data to detect mycoplasma by PCR, indicating that there is a correlation between ratios greater than 1 and detection of mycoplasma DNA. This is shown in FIG. 2, where the positive PCR band on the gel correlates with a ratio greater than 1.

Use of the method of the invention in a process for removing mycoplasma contamination from a cell culture.
The inventor has also shown that when cells are treated with a representative mycoplasma scavenger (ICN-Flow), a quinolone antibiotic derivative, we can detect a decrease in the ratio B / A.

  The manufacturer (ICN-Flow) encourages 7 days treatment of cells in quarantine to ensure complete removal of contaminating mycoplasma. However, ratio data obtained using the assay method of the present invention showed that 7 days was not sufficient. This was evident from the fact that these ratios were greater than 1. Further, after removal of the treatment agent and continuous culture, the ratio increased and after the PCR test (Stratagene kit), the culture was again positive. These data are shown in Figure 3, where three different cell lines were found to be contaminated with M. hyorhinis.

  K562 and U937 cells are floating cell lines, but A549 cells are adherent cell types; therefore, these data confirm that the assay can be used for both adherent and floating cell types . This is also shown in FIG. 2, where CHO and COS-7 cells are adherent cell types commonly used in cell culture laboratories.

  FIG. 3 also shows 10 days of treatment with MRA, and COS-7 and CHO cell cultures were sufficient to remove contaminating mycoplasma.

Bacterial filter failure to exclude mycoplasmas We also show that culture supernatants that passed through several bacterial blocking filters continued to show a positive ratio indicating the presence of viable mycoplasma . This is shown in FIG.

  Mycoplasma can form colonies as large as 600 μm in diameter, but can also exist as single cells as small as 0.15 μm during its life cycle. Because of its small size, mycoplasma can pass through 0.45 μm and 0.22 μm filters commonly used to “sterilize” tissue culture reagents. Figure 4 also confirms that the assay can be performed in the presence of cells, but that there is low detection sensitivity. Therefore, the assay method of the present invention is preferably performed on a sample substantially free of cells. This can be easily accomplished by centrifuging the cell culture, sampling the supernatant, and optionally filtering through a bacterial filter.

Preferred Assay Sensitivity As shown in FIGS. 5 and 8, dilution of the supernatant shows assay sensitivity, in which a 1: 1000 dilution of contaminated culture supernatant still gives a ratio greater than 1 be able to. Depending on the specific activity of acetate kinase and carbamate enzymes of different Mollicutes species, the sample can be further diluted. The dilution range will also vary depending on the number of colony forming units in the test sample.

Variations on the Assay Method of the Invention The assay method of the invention works without external addition of carbamate and acetate kinase substrates, carbamoyl phosphate and acetyl phosphate or precursors thereof in the form of ADP. I will. In contaminated culture samples, acetyl phosphate and carbamoyl phosphate or precursors thereof are endogenously present with sufficient cellular ADP from the cell culture to stimulate a response towards the formation of ATP. Let's go. Alternatively, ADP can be generated by other exogenously added enzymes or cellular enzymes, ie, adenylate kinase utilizing ATP and AMP.

  You can have a system that avoids adding these substrates directly and produces these substrates themselves. By using acetate and ammonia with ATP, the acetate kinase and carbamate kinase enzymes can produce acetyl phosphate and carbamoyl phosphate, which can then be used by the same enzymes to produce ATP from ADP. .

  The two substrates synthesize acetyl phosphate and carbamoyl phosphate, respectively, by utilizing the first part of the glucose fermentation and arginine lysis pathways, adding for example acetyl CoA and citrulline which can be used by mycoplasma enzymes Thus, it can be produced from a “precursor”.

  The following figure shows the difference between the biochemical activities of M. fermentans, which preferentially produces ATP via the carbamate kinase pathway, but will also utilize the acetate kinase pathway. FIG. 6 shows the effect of adding substrate individually for the enzyme pathway and then in combination with reagents.

  M. fermentans utilizes both pathways, M. orale utilizes only the carbamate kinase pathway, and positive ratios are observed only in the carbamate reagents alone or in combination as shown in FIG. FIG. 8 shows that the detection limit for M. orale is as low as 14 CFU / well.

  The inventor is investigating a mycoplasma that preferentially utilizes the acetate kinase pathway, namely M. hyorhinis. The data is shown in FIG.

  The inventor has tested over 15 different cell lines (see Table 3) and none of these cells has sufficient basic enzyme activity to affect the ratio and give false positives. Showed that. The inventor, without wishing to be bound by theory, believes that this is because these pathways are anaerobic and all mammalian cell cultures produce ATP by oxidative phosphorylation. . Therefore, by using only carbamoyl phosphate or its precursor, or only acetyl phosphate or its precursor, the mycoplasma contamination in question comes from the group that uses the acetate kinase pathway, the carbamate kinase pathway, or both An assay of the present invention can be generated that can be determined. This may have useful diagnostic applications.

  Bacteria with acetate kinase activity are not limited to those normally found as contaminants in cell cultures, but there may be exceptions for certain E. coli species that are handled in laboratories primarily for molecular biology. However, activity on this microorganism is only seen at very high inoculum concentrations, where there is an increase in turbidity and the resulting turbidity of the sample is easily observed by the naked eye. Therefore, if necessary, the method of the invention can be modified to include an initial screening step for bacterial contamination. This can be accomplished by a variety of methods, but is preferably done by passing the sample through a standard bacterial filter (Baseman and Tully, 1997).

Preferred reagent elements for use in the mycoplasma assays and kits of the invention
1.Mycoplasma detection reagent (MDR) per 100ml ・ Magnesium acetate 214.5mg (10mM)
・ Inorganic pyrophosphate 178.4μg (4μM)
・ Bovine serum albumin 160mg (0.16%)
・ D-Luciferin 10mg (360μM)
・ L-luciferin 250μg (8.9μM)
・ Luciferase (RY) 85μg
・ ADP 250.5μg (5μM)
・ AMP 69.44mg (2mM)
* RY is the name given to the recombinant luciferase supplied by Lucigen. A mixture of D-luciferin and L-luciferin has been found to give a more stable light output than D-luciferin alone.

2.Mycoplasma substrate (MS), per 100 ml ・ Acetyl phosphate 55.23 mg (3 mM)
・ Carbamoyl phosphate 45.87mg (3mM)

Examples of reactions that produce mycoplasma substrate (MS) precursors acetyl phosphate or carbamoyl phosphate:

Supplier
Sigma-Aldrich Company Ltd.
Fancy road
Poole
Dorset
BH12 4QH
UK

3. Buffer for Mycoplasma Assay (MAB), per 100ml ・ HEPES 1.1915g (50mM)
・ EDTA 74.44mg (2mM)
・ Triton X-100 250μl (0.25%)
・ PH7.50

Preferred concentration ranges of elements for use in the mycoplasma assay methods and kits of the present invention Preferred concentration ranges include:
ADP, 1 μM to 100 mM, preferably 1 μM to 100 μM, more preferably 5 μM.
AMP, 1 μM to 100 mM, preferably 0.1 mM to 10 mM, more preferably 2 mM.
Acetyl phosphate, 1 μM to 100 mM, preferably 0.1 mM to 20 mM, more preferably 3 mM. Concentrations above 10 reduce light output, but the assay is still effective.
Carbamoyl phosphate, 1 μM to 100 mM, preferably 0.1 mM to 20 mM, more preferably 3 mM.

Effect of surfactants on mycoplasma assay Disrupting the cell membrane of viable mycoplasma to allow release of the enzyme into the sample is a preferred embodiment of the assay method of the invention.
This allows for substrate binding and ATP generation. However, a positive ratio indicative of mycoplasma contamination can be obtained in the absence of any lysis treatment. This is indicated by the possibility that these enzymes can be released by viable mycoplasmas. It is also possible that some dead microorganisms released their contents by natural lysis.

  The addition of a lower concentration of the nonionic surfactant Triton-X100 greatly increases the sensitivity of the assay by ensuring maximum release of carbamate kinase and acetate kinase into the sample.

  The purpose of the following experiment was to determine the concentration of Triton-X100 in Hepes-EDTA buffer, with respect to the ratio found for mycoplasma contaminated K562 cell cultures. Two microorganisms, M. hyorhinis and M. orale, were examined.

  FIG. 10 shows that the mycoplasma enzyme can be detected without a lysis step with a surfactant. FIG. 10 also shows a decrease in light output at concentrations greater than 4-5%, which is due to the adverse effect of the surfactant on the luciferase enzyme / reaction. However, positive activity can also be detected at concentrations as high as 20% (v / v).

  The inventor has also shown that there was no detectable carbamate kinase or acetate kinase activity from E. coli strain JM109 cells at the concentration of Triton-X100 used in previous experiments.

  The above results confirm that there is no positive ratio for the number of bacterial cells used. Increasing the Triton-X100 concentration to levels reported to lyse bacterial cells (1-2%) still did not result in positive ratios greater than 1.

  In general, biological surfactants are commonly used to break up and release lipid bipolar membranes and then dissolve membrane-bound proteins. Nonionic surfactants are non-denaturing and can dissolve membranes without compromising biological activity. Nonionic surfactants are mainly used for studying protein conformation and for separating hydrophilic proteins from membranes on which hydrophobic proteins are spread. Anionic and cationic surfactants result in large modifications of the protein structure and are more effective in breaking protein aggregates. Zwitterionic surfactants are also less denatured but are effective in breaking protein aggregates.

  In order to efficiently lyse, release and preserve both eukaryotic and prokaryotic protein elements, these different groups of surfactants have been studied with several different cell types.

  For the preferred assays of the invention, the required lysing agent is a lysing agent that causes the mycoplasma membrane to break and allows the release of metabolic enzymes required to react with the substrate. Since there is no detergent removal or neutralization step, it is therefore important that the system chosen does not harm the activity of carbamate kinase and / or acetate kinase, or the luciferase / luciferin / ATP reaction. It is also preferred to use a system that selectively causes lysis of mycoplasma with little or no effect on bacteria that may be a potential source of cell culture / sample.

  However, the presence of a filtration step with a 0.45 μm filter should remove any source of larger microorganisms.

  An important difference between bacteria and mycoplasma is the lack of a cell wall, which makes it more difficult to lyse the bacteria. There are several very rigorous methods that can lead to overall dissolution, including pressure (French Press) and sonication. Other enzymatic digestion methods include the addition of lysosomes and then a surfactant. However, mycoplasma can be dissolved at a Triton X-100 concentration of about 1-2%.

  Low concentrations of other nonionic surfactants such as Brij® 35 (0.4%) (Sigma-Aldrich Company Ltd.) and B-PER (1%) (Perbio Science UK Ltd.) are luciferases The mycoplasma membrane can be destroyed without adversely affecting the enzyme and without adversely affecting the luciferase reaction. The concentration of these surfactants can be increased to 10% without losing the sensitivity of mycoplasma detection.

  Contaminating mycoplasma can be detected without a dissolution step to destroy the mycoplasma film. However, adding a light lysis step (0.25% Triton X-100 in Hepes-EDTA buffer) increases the sensitivity of the assay by releasing the mycoplasma enzyme into the reaction mixture.

  The lysis step preferably causes selective lysis of mycoplasma, but seems to have little or no effect on bacterial cells. A low concentration of most nonionic surfactants should do this. However, the filtration step physically removes any contaminating bacteria and allows the use of any detergent, but preferably does not inhibit the luciferase reaction or carbamate kinase and acetate kinase activities. Seem.

Preferred kit contents
LT07-118 (sufficient for 10 tests)
1.LT27-217, mycoplasma detection reagent, lyophilized state. 2 x 600 μl vial.
2. LT27-218, buffer for mycoplasma assay, 1 x 10 ml bottle.
3. LT27-221, mycoplasma substrate, lyophilized state. 2 x 600 μl vial.

LT07-218 (sufficient for 25 tests)
1.LT27-217, mycoplasma detection reagent, lyophilized state. 5 x 600 μl vial.
2. LT27-218, buffer for mycoplasma assay, 1 x 10 ml bottle.
3. LT27-221, mycoplasma substrate, lyophilized state. 5 x 600 μl vial

LT07-318 (sufficient for 100 tests)
1.LT27-216, mycoplasma detection reagent, lyophilized state. 1 x 10 ml vial.
2. LT27-220, buffer for mycoplasma assay, 1 x 20ml bottle.
3. LT27-224, mycoplasma substrate, lyophilized state. 1 x 10ml vial

Preferred reagent compositions for the kits and methods of the invention
1.Mycoplasma detection reagent (MDR) per 100ml ・ Magnesium acetate ¹ 214.5mg (10mM)
Inorganic pyrophosphate ¹ 178.4μg (4μM)
・ Bovine serum albumin ¹ 160 mg (0.16%)
・ D-Luciferin ² 10mg (360μM)
・ D-Luciferin ² 250μg (8.9μM)
・ Luciferase (RY) ³ 85μg
・ ADP ¹ 250.5μg (5μM)
・ AMP ¹ 69.44mg (2mM)

2.Mycoplasma substrate (MS), per 100 ml ・ Acetyl phosphate ¹ 55.23 mg (3 mM)
・ Carbamoyl phosphate ¹ 45.87 mg (3 mM)

3. Buffer for Mycoplasma Assay (MAB) per 100mlHEPES ¹ 1.1915g (50mM)
・ EDTA ¹ 74.44mg (2mM)
・ Triton X-100 ¹ 250μl (0.25%)
・ PH7.50

Preferred concentration range:
ADP, 1 μM to 100 mM
・ AMP, 1μM-100mM
Acetyl phosphate, 1 μM to 100 mM, preferably 1 mM to 10 mM
・ Carbamoyl phosphate, 1 μM to 100 mM

Supplier
1.
Sigma-Aldrich Company Ltd.
Fancy road
Poole
Dorset
BH12 4QH
UK
2.
Resem BV
Goudenregenstraat 84
NL-4131 BE Vanen
Netherlands
3.
Lucigen Ltd
Porton down science park
Porton, Salisbury
Wiltshire SP4 OJQ
UK

  Preferred embodiments of the present invention provide selective biochemical tests that utilize the activity of several mycoplasma enzymes. The presence of these enzymes provides a rapid screening procedure and enables sensitive detection of contaminating mycoplasma in the test sample. Viable mycoplasma is dissolved and the enzyme reacts with a mycoplasma substrate that catalyzes the conversion of ADP to ATP.

  By measuring the level of ATP in the sample before (A) and after (B) addition of the mycoplasma substrate, a ratio B / A indicating the presence or absence of mycoplasma can be obtained. In the absence of these enzymes, the second reading does not show an increase over the first reading (A), while higher due to the reaction of mycoplasma enzymes with their specific substrate in the mycoplasma substrate reagent. ATP level is provided. This increase in ATP can be detected using the following bioluminescent reaction.

  The intensity of the emitted light is directly related to the ATP concentration and is measured using a luminometer. The assay is preferably performed at ambient room temperature (18-22 ° C.) and at the optimum temperature for luciferase activity.

Method Overview It is preferred to centrifuge the culture supernatant to remove cells and, in some cases, pass through a bacterial filter prior to performing the assay.
The kit contains all the reagents required to perform the assay.
100 μl of culture supernatant is taken as a sample.
Add mycoplasma detection reagent (MDR).
Wait 5 minutes.
Read luminescence (A).
Add mycoplasma substrate (MS).
Wait 10 minutes.
Read luminescence (B).

Reagent reduction and storage Ensure that you follow the exact reagent reduction applicable to the relevant kit (10, 25 or 100 assay points).
This procedure usually requires an equilibration time of at least 15 minutes.
The mycoplasma detection reagent (MDR) and the mycoplasma substrate (MS) are preferably supplied as lyophilized pellets. These are reconstituted in Mycoplasma Assay Buffer (MAB) to produce a working solution for use in the assay.

For 10 tests (kit LT07-118):
1. Preparation of mycoplasma detection reagent
Add 600 μl mycoplasma assay buffer to the vial containing the lyophilized mycoplasma detection reagent.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

2. Preparation of mycoplasma substrate
Add 600 μl mycoplasma assay buffer to the vial containing the lyophilized mycoplasma substrate.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

3. Mycoplasma assay buffer This is preferably provided ready for use. Store at 2-8 ° C when not in use.

For 25 tests (kit LT07-218)
1. Preparation of mycoplasma detection reagent
Add 600 μl mycoplasma assay buffer to the vial containing the lyophilized mycoplasma detection reagent.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

2. Preparation of mycoplasma substrate
Add 600 μl mycoplasma assay buffer to the vial containing the lyophilized mycoplasma substrate.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

3. Mycoplasma assay buffer This is preferably provided ready for use. Store at 2-8 ° C when not in use.

For 100 tests (kit LT07-318)
1. Preparation of mycoplasma detection reagent
Add 10 ml mycoplasma assay buffer to the vial containing the lyophilized mycoplasma detection reagent.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

2. Preparation of mycoplasma substrate
Add 600 μl mycoplasma assay buffer to the vial containing the lyophilized mycoplasma substrate.
Replace cap and mix gently.
Allow the reagents to equilibrate for 15 minutes at room temperature.

3. Mycoplasma assay buffer This is preferably provided ready for use. Store at 2-8 ° C when not in use.

apparatus
1. Equipment This kit requires the use of a luminometer. Evaluate luminometer parameters and use less conditions to produce the correct programming of the machine.

The preferred assay of the present invention was designed for use with a cuvette / tubular luminometer. For use with plate illuminometers, see below.
Cuvette / Tubular luminometer:
Read time 1 second (accumulated value).

2. Other equipment and consumables
a.10ml sterile pipette
b. Luminometer cuvette
c.Micro pipettor -50 ~ 200μL; 200 ~ 1000μl
d. Desktop centrifuge

Preferred Test Protocol It should be noted that the culture medium sample should be taken prior to any other processing step, eg, trypsinization.
1. Bring all reagents to room temperature before use.
2. Replace mycoplasma detection reagent and mycoplasma substrate in mycoplasma assay buffer. Leave at room temperature for 15 minutes to ensure complete rehydration.
Transfer 3.2 ml of cell culture or culture supernatant to a centrifuge tube and pellet any cells at 1500 rpm (200 × g) for 5 minutes.
4. Transfer 100 μl of clear supernatant to luminometer cuvette / tube.
The luminometer is programmed to obtain an integrated reading of 5.1 seconds (this is usually the default setting for most cuvette luminometers).
6. Add 100 μl mycoplasma detection reagent to each sample and wait for 5 minutes.
7. Place the cuvette in the luminometer and start the program (Reading A).
8. Add 100 μl mycoplasma substrate to each sample and wait for 10 minutes.
9. Place the cuvette in the luminometer and start the program (Reading B).
10. Calculate the ratio = Reading B / Reading A.

Interpreting the results The ratio of reading B to reading A is used to determine if the cell culture is contaminated with mycoplasma.

  The speed and convenience afforded by the kits of the present invention means that the kits of the present invention provide a unique method for screening cultures for the presence of mycoplasma. Thus, the kit of the present invention is ideally suited for general testing of cells in culture. Frequent use of the test method of the present invention indicates that when a cell line is infected, it is prompted to take therapeutic action. The test method of the present invention can also be extended to complete cell transfer cell lines and commonly used components.

  Within each experimental situation, the interpretation of the different ratios obtained can vary depending on the cell type and conditions used. However, this test gives a ratio B / A of less than 1 for uninfected cultures. Cells infected with mycoplasma will usually produce a ratio greater than 1.

Protocol for plate illuminometer
1. Bring all reagents to room temperature before use.
2. Replace mycoplasma detection reagent and mycoplasma substrate in mycoplasma assay buffer. Leave at room temperature for 15 minutes to ensure complete rehydration.
Transfer the 3.2 ml cell culture supernatant cell culture to a centrifuge tube and pellet any cells at 1500 rpm (200 × g) for 5 minutes.
4. Transfer 100 μl of the clear supernatant to a luminescent compatible plate.
Program the luminometer to obtain an integrated reading of 5.1 seconds.
6. Add 100 μl mycoplasma detection reagent to each sample and wait for 5 minutes.
7. Place the plate in the luminometer and start the program (Reading A).
8. Add 100 μl mycoplasma substrate to each sample and wait for 10 minutes.
9. Place the plate in the luminometer and start the program (Reading B).
10. Calculate the ratio = Reading B / Reading A.

  Great care must be taken when handling any of the reagents. The skin has a high level of ATP on its surface, which can contaminate the reagents and give false high readings. Latex gloves avoid this problem.

  The optimum working temperature for all reagents is 22 ° C. If the reagent is frozen, always allow time for the reagent to reach room temperature (18-22 ° C) before use.

  Hidden contamination can be detected by the sensitivity of the assay, and if this ratio barely exceeds 1 (eg up to 1.3), it is recommended to retest the sample. Any culture that has been kept in quarantine can be tested after an additional 24-48 hours of culture to see if the ratio has increased.

Overview The assay of the present invention can be performed in the presence or absence of cells. Unlike known mycoplasma detection systems, the assay of the present invention can quickly screen samples using an inexpensive manual luminometer system, giving results within 15 minutes and proper handling of contaminated samples Can be made possible.

  PCR and DAPI / Hoechst stain will bind to all DNA from live or dead mycoplasma. Therefore, when examining the processing and removal of mycoplasma, even if the mycoplasma has been eradicated, the use of a PCR / DNA stain can still result in false positives.

  The assay of the present invention can detect viable mycoplasmas, whereas known methods such as PCR cannot distinguish between live and dead mycoplasmas.

[References]

FIG. 2 is a diagram of the kinetics of ATP generation in the presence of M.hyorhinis contamination. FIG. 4 is a comparison between the PCR kit from Stratagene and the ratio of a preferred embodiment of the present invention. FIG. 2 is a diagram of processing a contaminated cell line using the mycoplasma removal material of a preferred embodiment of the invention. FIG. 4 is a ratio data chart for cells, supernatant and supernatant filtered through 0.45 μm (F1), 0.22 μm (F2) and 0.1 μm (F3) filters. It is a figure of the influence of dilution of a supernatant liquid. Fig. 5 is a diagram of M. fermentans at 7900 CFU / well tested against different substrates. Figure 2 is a diagram of M. orale stock at 1450 CFU / well, tested against different substrates. FIG. 5 is a dilution diagram of M. orale stock to show the sensitivity of the assay. M. hyorhinis, a comparison of different substrate reagents. FIG. 6 is a diagram of the effect of Triton-X100 concentration on the detection of mycoplasma enzyme activity in K562 cells infected with M. hyorhinis (MH) and M. orale (MO). FIG. 7 shows the effect of high Triton-X100 concentration on K562 cells contaminated with M. hyorhinis (MH) compared to a large number (1-10,000 cells / 100 μl sample) of bacterial cells (E. coli).

Claims (39)

A method for detecting the presence of contaminating mycoplasma in a test sample comprising:
(i) Prepare a test sample that does not know whether mycoplasma exists ,
(ii) detecting and / or measuring the activity (B) of acetate kinase and / or carbamate kinase in said test sample, said activity indicating the presence of contaminating mycoplasma; and
(iii) confirming that the test sample is contaminated with mycoplasma based on the detection and / or measurement of the activity in step (ii). The following steps performed after step (ii) but before step (iii):
(iia) obtaining information (A) of acetate kinase and / or carbamate kinase activity detected and / or measured in a corresponding control sample; and
(iib) further comprising the step of comparing the activity (B) detected and / or measured in the test sample with the activity (A) in the control sample, the activity detected and / or measured in the test sample in step (ii) If (B) is greater than the activity (A) in the control sample in step (iia), i.e. if the ratio B / A is greater than 1, the test sample is contaminated with mycoplasma in step (iii) Check,
The method of claim 1. Detection and / or measurement of acetate kinase and / or carbamate kinase activity (B) in said test sample in step (ii) and / or acetate kinase and / or carbamate kinase in corresponding control sample in step (iia) Obtaining activity information (A) results in the following reactions:

A method according to claim 1 or 2, comprising detecting and / or measuring the appearance and / or disappearance of one or more substrates and / or one or more products.   Treating the test sample with a mycoplasma lysing agent to release mycoplasma cell contents into the sample after step (i) but further before step (ii). Item 4. The method according to any one of Items 1 to 3.   5. A method according to claim 4, wherein the solubilizer is a surfactant.   6. The method according to claim 5, wherein the lysis treatment with the surfactant cannot lyse bacterial cells.   The method according to any of claims 4 to 6, wherein the corresponding control sample is the same as the test sample before the mycoplasma lysis treatment.   The corresponding control sample is the same as the test sample, but obtaining detection / measurement information regarding the test sample activity occurs after a certain time interval after obtaining detection / measurement information about the control sample. 5. The method according to any one of 2 to 4. Said time interval is 3 0 minutes and less, the method according to claim 8.   10. The method according to any of claims 1 to 9, wherein the detecting and / or measuring step comprises detecting and / or measuring ATP.   The method according to claim 10, wherein the ATP is detected and / or measured by a luminescent reaction.   12. The method according to claim 11, wherein the luminescent reaction is a bioluminescent reaction.   13. A method according to any one of the preceding claims, wherein ADP is added to the test sample prior to the detection and / or measurement step (ii).   Mycoplasma substrate (MS) reagent is added to the test sample prior to the detection and / or measurement step (ii), and the MS reagent contains acetyl phosphate or precursor thereof, and / or carbamoyl phosphate or precursor thereof. 14. The method according to any one of claims 1 to 13, comprising.   15. The method according to claim 14, wherein the precursor of acetyl phosphate is acetyl CoA.   15. The method of claim 14, wherein the carbamoyl phosphate precursor is citrulline and / or ammonia.   17. A method according to any of claims 13 to 16, wherein the control sample is the whole or an amount of the test sample to which no mycoplasma reagent has been added.   18. A method according to any of claims 2 to 7, or 9 to 17, wherein the control sample has been shown to be free of mycoplasma by another method.   19. The method of claim 18, wherein the control sample has been shown to be free of mycoplasma by one or more of PCR testing, DNA fluorescent staining or mycoplasma culture methods.   20. The method according to any one of claims 1 to 19, wherein the test sample and / or control sample is a cell culture sample.   The cells in the cell culture sample are mammalian cells, preferably Vero, MRC5, HUVEC, BSMC, NHEK, MCF-7, AoSMC, A549, HepG2, FM3A, PC12, ARPE-19, CHO and COS cells, etc. 21. The method of claim 20, wherein said adherent cells and / or primary adherent cells isolated from animal sources.   22. the method of.   21. The method of claim 20, wherein the cell culture is a plant cell culture.   24. A method according to any of claims 20 to 23, wherein the cell culture sample is a sample derived from a cell culture but which is itself substantially free of cellular material.   20. The method according to any one of claims 1 to 19, wherein the test sample and / or control sample consists of a cell-free reagent.   26. The method of claim 25, wherein the cell-free reagent is trypsin.   A method for treating a cell culture to remove mycoplasma contamination, wherein the mycoplasma contaminated cell culture is treated with an agent to remove and / or destroy the mycoplasma, then claim 1. To test a sample from the culture for mycoplasma contamination using the method according to any one of 1 to 26, and if necessary, the process is repeated until no mycoplasma contamination is detected in the sample. A method comprising repeating one or more times. A method for detecting the presence of mycoplasma in a test sample comprising the following steps:
(i) preparing a test sample that does not know whether mycoplasma exists ,
(ii) without adding an exogenous reagent to convert ADP to ATP, a bioluminescent reaction is used to detect or measure ATP in the test sample to measure ATP and / or light output (A Detecting and / or measuring the activity of acetate kinase and / or carbamate kinase in said test sample by obtaining
(iii) obtaining a measurement (B) of ATP and / or light output from the corresponding control sample;
(iv) comparing the ratio B / A of the measured values of ATP and / or light output; and
(v) confirming that the test sample is contaminated with mycoplasma if the ratio B / A is greater than 1;   29. A method according to any one of claims 1 to 28, comprising passing the test sample through a filter holding bacterial cells.   30. Use of acetyl phosphate or a precursor thereof and / or carbamoyl phosphate or a precursor thereof in a method for detecting contamination of a sample by mycoplasma as defined in claims 14-29. A kit for use in detecting mycoplasma contamination, including:
(i) acetyl phosphate or precursor thereof, and / or carbamoyl phosphate or precursor thereof,
(ii) an excessive amount of ADP to drive the enzymatic reaction in the direction of ATP formation,
(iii) A kit comprising one or more agents for dissolving mycoplasma.   32. The kit of claim 31, wherein the agent for dissolving mycoplasma comprises a surfactant.   33. The kit according to any one of claims 31 to 32, further comprising means for detecting and / or measuring ATP by a luminescent reaction.   34. The kit of claim 33, wherein the means comprises a mycoplasma detection reagent (MDR) including magnesium acetate, inorganic pyrophosphate, bovine serum albumin, luciferin luciferase, ADP and AMP.   35. The kit according to any one of claims 31 to 34, wherein the reagent is provided in a lyophilized state.   36. The kit according to any one of claims 31 to 35, further comprising a mycoplasma assay buffer (MAB) capable of returning the lyophilized reagent to its original state. The buffer maintains the pH of 7.5, The kit of claim 36.   35. Kit according to claim 33 or 34, further comprising a luminometer, preferably a manual luminometer.   39. The kit according to any one of claims 31 to 38, further comprising a bacterial filter.

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