BRPI0407750B1 - method for detecting the presence of mycoplasma in a test sample, use of acetyl phosphate or a precursor thereof and / or carbamoyl phosphate or a precursor thereof, and kit for use in detecting mycoplasma contamination - Google Patents

Abstract

"method for detecting the presence of mycoplasma in a test sample, process for treating a cell culture to remove mycoplasma contamination, use of acetyl phosphate or a precursor and / or carbamoyl phosphate or a precursor thereof, and, kit for use in detecting mycoplasma contamination ". The invention relates to a method for detecting the presence of mycoplasma in a test sample, comprising: (i) providing a test sample and (ii) detecting and / or measuring acetate kinase and / or carbamate kinase activity in the sample test activity indicating mycoplasma contamination.

Description

“METHOD FOR DETECTING THE PRESENCE OF M1COPLASM IN A TEST SAMPLE, USE OF ACETILA Phosphate OR A PRECURSOR AND / OR CARBAMOIL Phosphate, AND, KIT FOR USE IN THE DETECTION OF CONTAMINATION BY THE PRESENT INVENTION regarding test methods and materials for detecting members of the Mollicutes family that contaminate a test sample, such as a sample from a cell culture.

Taxonomically, the lack of cell walls has been used to separate Mollicutes from other bacteria in a class called Mollicutes {Razin et al. 1998). Members of this class are summarized in the following Table 1. TABLE 1 MAIN CHARACTERISTICS AND TAXONOMY OF CLASS MOLLICUTES

In the context of this application, the term “mycoplasma” is intended to cover all members of the Mollicutes class, not just Mycoplasmatales. In fact, "mycoplasma" is the common term in the technique for all Mollicutes.

Mycoplasmas are dispersed in nature as parasites of humans, mammals, reptiles, fish, arthropods and plants. They are the smallest and simplest prokaryotes. They lack a rigid cell wall and are incapable of peptidoglycan synthesis; they are therefore not sensitive to antibiotics such as penicillin and its analogs. Mycoplasmas developed by degenerate evolution of Gram-positive bacteria with a low molecular percentage of DNA guanine and cytosine content, ie Lactobaállus, Bacillus, Streptococcus and two Closiridium species. Mollicutes lost, during the process of evolution, a substantial part of their genetic information. It is this limited coding capacity that has imposed the need for a parasitic way of life. Most species are facultative anaerobic, but some are obligatory and hence the similarities in their metabolism to anaerobic bacteria.

More than 180 Mollicute species have been identified, of which 20 distinct Mycoplasma and Acholeplasma species from humans, cattle and pigs have been isolated from cell culture. 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 of other cell lines introduced into laboratories. Also an undesirable source of exogenous mycoplasma can be found in tissue culture reagents such as serum products. Mycoplasma contamination, unlike bacterial, rarely produces cloudy growth or obvious cell damage. Viable mycoplasma can be recovered from work surfaces seven days after inoculation, and mycoplasma can also pass through bacteria retention filters.

In its maximum population phase, there may be as many as 108 mycoplasmas / ml of supernatant, in a 5: 1 ratio to host cells. If present, mycoplasmas "grow" at detectable concentrations in the culture medium, they are then also adsorbed onto the cell surface. It is a moot point whether mycoplasma enters and survives within cultured mammalian cells.

Mycoplasmas are capable of altering almost all properties of an in vitro culture. They deplete crop nutrients, particularly arginine. Infected eukaryotic cells show aberrant growth, changes in metabolism and morphology. Certain biological properties have been implicated as determinants of virulence; These include secretion or introduction of mycoplasmic enzymes such as phospholipases, ATPases, hemolysins, proteases and nucleases into the host cellular environment.

A major problem with mycoplasma is that its contamination is often hidden and, unlike bacterial detection, cannot be easily visualized. Their resistance to antibiotics and their ability to pass through normal bacterial sterilization filters means that they can circumvent the typical precautions of cell culture technique. As a result of the negative impact of having these imperceptibly proceeding contaminations, it has become apparent that continuous screening is essential for any cell culture laboratory.

Several studies have shown that at least 10% to 15% of cultured cells may be contaminated with mycoplasma (Rottem and Barile 1993, McGarrity and Kotani 1985). Most cellular biologists recognize the need for routine testing for mycoplasma, but because of the cost and inaccuracies of the tests currently available, this has so far persisted as an unrealizable ideal. The only accurate method available for detecting viable mycoplasma is the culture of microorganisms. However, the difficulty associated with its in vitro culture has proven to be problematic because of the complex media required for its cultivation (Razin et al. 1998). Culture has also been considered to be the most sensitive method as it is said to be able to detect a single viable organism. However, the results take two to three weeks by highly skilled personnel with very specific culture requirements. The time consumed is a result of the need to cultivate the cells to a sufficient number by which they form colonies, which can then be evidenced by using a Dienos spot. Mycoplasma can be cultured on agar and in broth culture, with most mycoplasmas producing microscopic colonies with a characteristic 'stellate egg' appearance, growing implanted in the agar, although some colonies may not grow fully implanted. There are some strains that cannot be easily grown using the broth agar or medium. These strains require cell-assisted culture for isolation and identification. This last approach helps in the identification and detection of mycoplasma species adsorb on host cell surfaces (Rottem and Barile 1993). However, because of the complicated nature of culture procedures, these tests are very commonly done by mycoplasma testing service laboratories.

One of the simplest ways to detect mycoplasmas in samples is fluorochrome DNA assay. One of the most commonly used is 4 ', 6-diamine-2-phenylindole dihydrochloride (DAPI), but Hoechst staining is considered to be the method of choice. Cell culture samples are taken, fixed and stained with Hoechst 33258 (bisbenzamide) and examined under UV epifluorescence (Battaglia et al. 1994, Raab 1999). If there are mycoplasmas associated with the cells, then the nuclei will appear surrounded by fluorescent structures in the cytoplasm. Negative cells are represented by nuclear staining of cellular DNA only. Accurate interpretation of DNA staining results requires experienced vision, and also requires specialized equipment, that is, a fluorescence microscope. Mycoplasma detection by PCR is a test commonly used by external service laboratories, and is also performed in those laboratories that have the appropriate equipment. Primers used in mycoplasma PCR kits associate with conserved regions of the mycoplasma genome, enabling the detection of several species (Raab 1999). The most commercially available PCR kits require amplified products to be analyzed by agarose gel electrophoresis, with the resulting band patterns determining the contaminant species present. However, visualization of band patterns is subjective. Roche Mycoplasma PCR ELISA (Raab 1999) has a different system and cannot distinguish between species. This kit includes digoxigenin-dUTP, and the PCR product is captured on the surface of the reservoirs on a microtiter plate coated with the anti-digoxigenin-peroxidase conjugate. Tetramethylbenzidine (TMB) stained product is observed using a standard ELISA plate reader. Life Technologies has developed the MYCOTECT® Kit, based on adenosine phosphorylase activity, which is found only in small amounts (if any) in mammalian cells (Verhoef et al. 1983). This enzyme converts 6-methylpurine deoxyriboside (6-MPDR) into two toxic products (6-methylpurine and 6-methylpurine riboside). The assay requires the addition of the contaminated cell line to an indicator cell line grown on a 24-well tissue culture plate. The 6-MPDR substrate is added and, after 3 to 4 days of further growth, a violet crystal stain is added to test for viability of the indicator cells, where mycoplasma positivity results in the production of these toxic agents. Although it has been reported to detect 1 mycoplasma cell per 200,000 target cells, if average conditions are adjusted to favor mycoplasma growth (Whitaker et al. 1987), the main disadvantage of this system is that it is labor intensive and time consuming. Mycoplasma antigens can be detected using immunoassays employing antibodies raised against mycoplasma antigens. For example, the detection of M. pneumoniae in clinical specimens (Daxboeck et al. 2003). The use of different antibodies takes into account species identification. There are several commercially available kits; for example IDEXX (US) laboratories provide enzyme linked immunosorbent assays (ELISA) for the detection of various mycoplasmas that have animal health implications. Most known tests take a minimum of 24 hours to complete, require expensive equipment and a significant amount of expertise. Likewise, they are strain specific assays. None of them are generic, ie they have the ability to detect mycoplasma species in general. UK Patent No. 2,357,336 B describes an assay that can be used to detect mycoplasmas in cell cultures. The assay is based on the observation that mycoplasmas overproduce the ATPase enzyme in large quantities. Mycoplasma ATPase activity transforms sufficient ATP into cellular or extremely added ADP to produce detectable ADP. Therefore, the assay is based on the detection of ADP and this is accomplished by adding an enzyme-containing reagent (containing a combination of pyruvate kinase and phosphoenol pyruvate; adenylate kinase; glycerol kinase, mykinase; or a combination) to the sample. creative phosphate), which turns ADP into ATP and detect ATP using a bioluminescent reaction. UK Patent Application No. 2,357,336 is incorporated herein, including for purposes of possible amendment. The present invention further seeks to provide means for detecting mycoplasmas in samples, such as cell culture samples.

In accordance with a first aspect, the invention provides a method for detecting the presence of contaminating mycoplasma in a test sample, comprising: (i) providing a test sample; (ii) detecting and / or measuring the activity (B) of acetate kinase and / or carbamate kinase in the test sample, and said activity being indicative of the presence of contaminating mycoplasma; and (iii) identifying the test sample when contaminated with mycoplasma based on detecting and / or measuring said activity from step (ii).

Preferably, the method further comprises the following steps performed after step (ii), but prior to 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) compare the activity detected or measured in the test sample (B) with that of the control sample (A); wherein the test sample is identified as contaminated with mycoplasma in step (iii) if activity (B) detected and / or measured in the test sample in step (ii) is greater than that of the control sample (A) in step (iia), that is, the ratio BI A is greater than one.

In a second aspect, the invention provides a method wherein detecting and / or measuring the activity (B) of acetate kinase and / or carbamate kinase in the test sample in step (ii) and / or obtaining the information (A) of Acetate kinase and / or carbamate kinase activity in a corresponding control sample in step (iia) comprises detecting and / or measuring the appearance and / or disappearance of one or more of the substrates and / or one or more of the products of the following reactions. Preferably, the step of detecting and / or measuring comprises detecting and / or measuring comprises detecting and or measuring ATP. Even more preferably, ATP is detected and / or measured by a light emitting reaction, especially a bioluminescent reaction.

Light-emitting systems have been known and isolated from many luminescent organisms, including certain bacteria, protozoa, coelenterates, mollusks, fish, millipedes, flies, fungi, worms, crustaceans and beetles, particularly the fireflies of the genera Photinus, Photuris and Luciola and kingfisher beetles of the genus pyrophorus. In many of these organisms, oxy-reductions take place in which free energy exchange is used to excite a molecule to a high energy state. Then, when the excited molecule spontaneously returns to the ground state, visible light is emitted. This emitted light is called “bioluminescence”.

Beetle luciferases, particularly that of the firefly species Photinus pyralis, have served as paradigms for understanding bioluminescence since earlier studies. P. pyralis luciferase is an enzyme that appears to have no tightly attached prosthetic groups or metal ions and has 550 amino acids and a molecular weight of about 60,000 daltons; The enzyme has been available in the art in crystalline form for many years. Studies of molecular components in the mechanism of firefly luciferases in producing bioluminescence have shown that the enzyme substrate is firefly luciferin, a polyheterocyclic organic acid, D - (-) - 2- ( 65- (hydroxy-2'-benzothiazolyl) Î ± -thiazoline-4-carboxylic acid (hereinafter referred to as "luciferin" unless otherwise indicated). ADP can be detected using the following bioluminescence reaction: The intensity of light emitted is linearly related to ATP concentration and is measured using a luminometer. Luciferase has been used as a means of assaying minute concentrations of ATP; As little as 10'16 moles of ATP can be detected with high quality enzyme preparations. The luciferase-luciferin reaction is highly ATP specific. For example, deoxy-ATP produces less than 2% of the light generated by ATP, and other triphosphate nucleosides produce less than 0.1%.

Crystalline luciferases can be isolated directly from the light organs of the beetles. CDNAs encoding the luciferases of various beetle species [including but not limited to P. pyralis luciferase (firefly), the four P. plagiophthalamus luciferase isozymes (salta-martim beetle), L. cruciata luciferase (firefly) and L. Lateralis luciferase] (from Wet et al., 1987, Masuda et al., 1989, Wood et al., 1989, European Patent Application Publication No. 0 353 464). available. In addition, cDNAs encoding the luciferases of any other beetle species producing luciferases are readily obtainable by the skilled artisan using known techniques (from Wet et al., 1986, Wood et al., 1989). With cDNA encoding a beetle luciferase available, it is entirely easy to prepare large amounts of the luciferase in highly pure form by isolating the bacteria (e.g., E. coli), yeast, cultured mammalian cells, or the like, which have been transformed to express cDNA.

In addition, the availability of cDNAs encoding beetle luciferases and the ability to rapidly screen for cDNAs encoding luciferase-luciferin reaction enzymes (see Wet et al., 1986, above, and Wood et al, above). They also allow the skilled person to prepare, and obtain in large quantities in pure form, the mutant luciferases that conserve activity in the bioluminescence catalyst production through the luciferase-luciferin reaction. Such a mutant luciferase will have an amino acid sequence that differs from the sequence of a naturally occurring beetle luciferase at one or more positions (White et al., 1996, WO 01/31028 and WO 00/24878). In the present specification, the term "luciferase" includes not only naturally occurring luciferases in beetles, but also mutants, which retain the activity of providing bioluminescence by catalyzing the luciferase-luciferin reaction of such naturally occurring luciferases. Most preferably, in the method of the invention, after step (i) but before step (ii), the sample is treated to lyse any mycoplasma and thereby release its cellular contents in the sample. Those skilled in the art will understand that lysis can be performed by a variety of methods, including the application of chemicals such as detergents, and mechanical methods such as sonication etc.

Advantageously, lysis is effected by treating the sample with a detergent, or other method of lysis, which takes into account the lysis of the Mycoplasma cell membrane, but does not affect the cell wall of any bacteria that may be present. Exemplary detergent treatment includes the use of low concentrations (e.g., 0.25% v / v) of a detergent such as Triton X100. The preferred lysis method is one that is sufficient to lyse the mycoplasmic membrane, but which should be ineffective against bacterial cells. In studies comparing eukaryotic cell lysis with bacterial lysis, it was observed that nonionic detergents (mainly polyethoxyethers) could be used to lyse somatic cells without affecting microbial cells (Schramm and Weyens-van Witzenberg 1989, Stanley 1989). It is the presence of the rigid cell wall that makes the bacteria less sensitive to detergent lysis, and stricter lysis procedures are required to lyse bacterial cells. For efficient lysis and total protein release, bacteria often require exposure to enzymes such as lysozyme to break the cell wall (Pellegrini et al, 1992). The most preferred conditions for mycoplasma detergent lysis are set forth below.

However, contamination with bacteria will produce cloudy growth, and the bacteria are also visible when viewing a cell culture under phase contrast microscopy. These bacterial cultures can be detected quite easily and discarded shortly thereafter.

Unlike bacteria, mycoplasma will pass through a 0.45 μΜ filter used for filter sterilization (Baseman and Tully 1997), and it is possible to distinguish between bacterial and mycoplasmic contamination by the addition of a filtration step.

Accordingly, in preferred embodiments of the invention, the test sample is passed through a bacterial filter in step (i). Of course, skilled people will note that if the test sample is treated to remove bacteria, for example by passing it through a bacteria-retaining filter, it is not important to selectively lyse mycoplasma, ie without lysing bacteria.

In a preferred embodiment, the ADP is added to the test sample prior to step (ii) of detecting and / or measuring. However, the assay may also use endogenous ADP.

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

By "a precursor thereof" has been included one or more compounds from which acetyl phosphate and / or carbamoyl phosphate may be generated. Exemplary reactions are outlined below: Therefore, instead of adding acetyl phosphate to the mycoplasma substrate reagent, a precursor such as acetyl-CoA may be included.

Similarly, instead of carbamoyl phosphate, a precursor such as citrulline and ammonia may be added to the mycoplasma substrate reagent. Most preferably, both acetyl phosphate and carbamoyl phosphate and / or their precursors are added to the sample prior to step (ii) in the methods of the invention. This allows a generic assay for mycoplasma contamination to be performed because mycoplasmas utilize either or both substrates via their acetate kinase and / or carbamate kinase enzymes.

Alternatively, a more specific assay can be produced using only one of the above substrates or their precursors. Such an assay will be specific for mycoplasma that only uses one of the acetate kinase or carbamate kinase enzymes. Table 2 below cites some examples of family members of Mollicutes (parasitic mammalian hosts) using acetate kinase preferably, carbamate kinase preferably, or both. In addition to those listed below, there are several of mycoplasmas that infect reptiles, insects, and plants where biochemical research has identified the use of these same pathways (Kirchoff et al. 1997, Forsyth et al. 1996, Taylor et al. 1996 and Tully et al. ah 1994). TABLE 2 ATP GENERATION BY MICROLASM THROUGH USING GLUCOSE OR ARGININE Most preferably, in all methods of all aspects of the invention, the "corresponding control sample" is the test sample prior to a mycoplasma lysis treatment. and / or adding a mycoplasma substrate and / or a time interval (e.g., more than approximately 30 minutes). In this preferred embodiment, both activity measurements are performed on the same sample, the test sample. A first measurement (A) of activity is taken before or concurrently with the mycoplasma lysis step, then, after the addition of a mycoplasma substrate and / or a time interval (eg, more than approximately 30 minutes), A second measurement (B) of the activity is taken. If the B / A value is greater than one, the test sample is identified as contaminated with mycoplasma.

Empowered persons will note that the "corresponding control sample" may also be a predetermined negative control sample, but this is less preferable.

In a preferred embodiment, the control sample the control sample must have been presented as being free of mycoplasma contamination. Suitable methods for doing so include PCR testing, fluorescent DNA staining, or culture methods as described in this report. Thus, in one embodiment, by "corresponding control sample" we mean a sample containing substantially the same material as that contained in the test sample, but which, unlike the test sample, has been found to be free of contamination by mycoplasma. Skilled people will note that an uncontaminated mycoplasma condition can be observed by a variety of known methods. Several suitable methods are examined by Rottem and Barile 1993, while a delineation 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, especially a mammalian cell culture. Some examples are listed in Table 3 below. TABLE 3 COMMONLY CULTIVATED CELL LINES WHICH HAVE BEEN TESTED WITH USE OF THE TEST METHOD .______________________________________ Cell Type Name Promoter / Cell Number ____________________________________________________ Deposit ________ K562 Human Chronic Myelogenous Leukemia ECACC 89121407 U837 H1101-8108 EC1501C1701701702708 EC1501C1701501501C1501501-6701 EC1501C1701-4701 7 Human Acute T-Lymphoblastic Leukemia ATCC CCL-119 Jurkat Human T-Cell Leukemia ECACC 88042903 CHO Chinese Hamster Ovary ECACC 85050302 COS-7 Transformed Simian Kidneys, EC40 ECCC 87021302 Vero Kidney Cells African MacC11115 Human Fetal Lung ECACC 84101801 HUVEC Human Umbilical Vein Endothelial Cells ECACC 89110702 BSMC Cambrex Human bronchial smooth muscle cells CC-2576 NHEK Normal Human Epidermal Keratinocytes Cambrex CC-2503 MCF-7 Adenocar CNS-8 Mama ECC12 Cambrex Aortic Smooth Muscle CC-2571 A549 Human Lung Carcinoma Cells ECACC 86012804 HepG2 Human Hepatocyte Carcinoma ECCC 85011430 FM3A Mouse Mammary Carcinoma ECACC 87100804 PC12 Human Cerebral Epithelial Epithelial Cellular Rheumatoid Cerebrospinal ECCC 87100804 PC12 2302 RT112 Human Bladder Carcinoma________________ECECC 85061106________ Where ECACC represents the European Collection of Animal Cell Culture, ATCC represents the American Tissue Culture Collection, and Cambrex represents Cambrex Bio Science Wokingham, United Kingdom.

Also, it should be possible to test primary mammalian cell types plus those cells maintained by the tissue banks, for example ATCC and ECACC. It is noteworthy that the assays of the invention can be used to detect mycoplasma contamination in cultures of both adherent cells (eg, HepG2, A549, CHO and COS cells) and cells that are cultured in suspension (e.g., Cells). Jurkats, U937, K562 and HL-60).

Preferably, the sample to be tested is from cell culture supernatant that has been previously centrifuged to remove cell material. However, it is also to perform the assay in the presence of cells or cellular debris.

Cell free samples can also be tested using the methods of the invention. For example, the methods of the invention are particularly useful for testing cell free reagent samples such as tissue culture media, and typically those containing animal derived materials such as serum (e.g. fetal bovine serum), trypsin. and other culture supplements, etc. Examples of some commonly used media and supplements that can be tested in this manner are given in Table 4. TABLE 4 TISSUE CULTURE MEANS AND SUPPLEMENTS THAT CAN BE TESTED BY USING THE TEST SYSTEM. A third aspect of the invention provides a method. detecting the presence of mycoplasma in a test sample, comprising the following steps: (i) providing a test sample; (ii) without adding an exogenous reagent (eg substrates for kinase activity) to transform ADP into ATP, detect or measure ATP in the test sample using a bioluminescent reaction to obtain ATP and / or light output measurement (A ); (iii) obtain an ATP and / or measure light output (B) from a corresponding control sample; (iv) compare the ratio of ATP and / or light output measurement j3 / A; and (v) identify the test sample as contaminated with mycoplasma in the event that the BI A ratio is greater than one.

As mentioned with respect to the above aspects of the invention, it is more preferable that mycoplasma lysis treatment and / or the addition of mycoplasma substrate and / or a time interval occur prior to step (ii).

As mentioned with respect to the foregoing aspects of the invention, it is more preferable that the "corresponding control sample" be the test sample, except that it has not undergone Mycoplasma lysis treatment and / or the addition of Mycoplasma substrate and / or left for a period of time (for example, more than approximately 30 minutes).

In other words, both measurements are taken from the test sample. Thus, in a preferred embodiment, control ATP and / or light output measurement is taken following addition to the detergent containing mycoplasma detection reagent sample and AMP-enhanced luciferase / luciferin, and Test ATP and / or light output measurement are taken following addition of substrates for kinase activity (or its precursors).

Another aspect of the invention provides an in vitro process for treating a cell culture for removing mycoplasma contamination comprising: treating a contaminated mycoplasma cell culture with an agent for removing or destroying mycoplasmas; and subsequently testing a culture sample for mycoplasma contamination using a method of the invention; if necessary, repeat the process one or more times until mycoplasma contamination is no longer detected in the sample.

Many of the routine antibiotics used in cell culture are ineffective against mycoplasmas. There are some agents that exhibit inhibitory activity, including gentamicin sulfate, kanamycin sulfate, and tylosin tartrate (www.unc.edu/depts/tcf/mycoplasma.htm). There are several commercial treatment products, including Mycoplasma Removal Agent (ICN-Flow), a derivative of quinolone family antibiotics, also a non-antibiotic treatment of Minerva Biolabs (Berlin, Germany), Mynox®. The American company Invivogen supplies Plasmocin®, which has two bactericidal components, one that acts on protein synthesis and the other that inhibits DNA replication. The antibiotics tetracycline and ciprofloxacin are reported to have success rates of less than 80 to 85% (www.unc.edu/depts/tcfmycoplasma.htm). It is therefore extremely difficult to completely eradicate mycoplasma from cultures once contamination has been established.

Many of the effective antibiotics are derived from quinolone, and the effectiveness of different antibiotics varies according to the species of mycoplasma being treated. Dufíy et al, 2000, investigated the viability of M. pneumoniae, M. hominis, M. fermentans, M. genitalium and U. urealyticum against quinolone gemifloxacin, and compared it with various antibiotics including tetracycline, clindamycin and other quinolones. . Results showed variable responses between species, but gemifloxacin performed better than tetracycline. There are some species that are resistant to tetracyclines because of the acquisition of the tetM gene. This is a frequent occurrence, and is complicated by variations in responses of species dependent on the source of mycoplasma. For example, mycoplasma exposed to antibiotics in eukaryotic cell culture have different profiles of the same species isolated from a human or animal source (Taylor-Robinson and Bebear 1997). Although the reported success of antimycoplasma treatments seems highly variable, a recent study by Uphoff et al. 2002 reports that 96% of leukemia lymphoma cell lines were taken free of mycoplasma with at least one of the treatments tested.

Examples incorporating the various aspects of the invention will now be described with reference to the accompanying figures in which: Figure 1: The kinetics of ATP generation in the presence of M. hyorhinis contamination.

Figure 2: A comparison between the Stratagene PCR Kit and a preferred embodiment of the inventive ratios.

Figure 3: Treatment of Mycoplasma Removing Agent contaminated cell lines according to a preferred embodiment of the invention.

Figure 4: Relationship data with cells, supernatants and supernatants filtered through 0.45 pm (F1), 0.22 pm (F2) 0.1 pm (F3) filters.

Figure 5: Effect of supernatant dilution.

Figure 6: M. fermentans at 7900 CFUs / well, tested against different substrates.

Figure 7: M. orale stock at 1450 CFUs / reservoir, tested against different substrates.

Figure 8: Dilution of M. orale stock to show assay sensitivity.

Figure 9: M. hyorhinis, comparison of different substrate reagents.

Figure 10: The effect of Triton-X100 concentrations on the detection of mycoplasma enzyme activities in M. hyorhinis (MH) and M. orale (MO) infected K562 cells.

Figure 11: Shows the effect of increasing Triton-X100 concentrations on M. hyorhinis (MH) -contaminated K562 cells compared to increasing numbers (1 to 10,000 cells / 100 μΐ of sample) of bacterial cells (E coli ).

EXAMPLE 1: TEST METHOD OF THE INVENTION The principle of the preferred test method is to provide the appropriate substrates for mycoplasmic enzymes. If mycoplasma contamination is present, there is a conversion of ADP to ATP, which can then be measured, preferably by the luciferase-luciferin reaction. Mycoplasma Detection Reagent is added and, after approximately 5 minutes, an initial light output reading (A) is taken, Mycoplasma Substrate (MS) is added and any enzymatic activity is allowed to progress for approximately 10 minutes, at which point a second light reading (B) is taken.

If there is mycoplasmic contamination then the second reading (B) will be higher when compared to the first reading (A) giving a B / A ratio of more than 1. If the culture is negative (uncontaminated by mycoplasma) then the ratio BI A will be 1 or, very often, smaller than one because of the drop in the luminescent light signal usually observed over time. Figure 1 demonstrates the reaction kinetics. Typically, the observed B / A ratio with mycoplasma contamination is much higher than 1; for example Figure 1 shows a ratio of 114.

A preferred assay kit of the invention comprises a Mycoplasma Detection Reagent (MDR); Mycoplasma Assay Buffer (MAB) for reconstitution of MDR and Mycoplasma Substrate (MS). MDR and MS are preferably provided as lyophilized preparations.

All mycoplasmas generate ATP either via the acetate kinase pathway or the carbamate kinase pathway. The Mycoplasma Substrate of the invention contains substrates for one or both of these enzymatic reactions. ADP is a requirement for both enzymes and is preferably supplied in excess in the Mycoplasma Detection Reagent of the invention to drive generation of ATP formation. MDR is added to a culture supernatant sample that has been previously centrifuged to remove cellular material, although the assay can be performed in the presence of cells. Alternatively, or additionally, the test sample may be passed through a bacterial filter. MDR contains substrates for luciferase, luciferin and other cofactors plus AMP and ADP. Mycoplasma Substrate (MS) contains carbamoyl phosphate and / or acetyl phosphate or their precursors, required for detection of carbamate and / or acetate kinases activities.

A preferred sample volume is 100 μΐ, to which 100 μΐ of reconstituted MDR is added. After approximately 5 minutes, the first luminometric reading (A) is taken, and it gives the base reading on which the other B / A ratio calculations are determined.

The assay methods of the invention were used to analyze contamination by Acholeplasma laidlawii, M. hyorhinis, M. fermentam, M. orale and M. genitalium and to detect various unknown mycoplasma contamination.

The inventors compared their data with PCR detection of mycoplasma, and showed that there is a correlation between ratios greater than one and mycoplasmic DNA detection. This is shown in Figure 2, where gel-positive PCR bands correlate with ratios of more than one.

EXAMPLE 2: USE OF INVENTION METHODS IN A PROCESS TO REMOVE MICROLASM CONTAMINATION OF A CELL CULTURE.

The inventors have also shown that we can detect a reduction in B / A ratios when cells are treated with an exemplary Mycoplasma Removal Agent (ICN-Flow), a derivative of the quinolone family of antibiotics.

The manufacturers (ICN-Flow) recommend 7-day treatment of quarantined cells to ensure complete removal of contaminating mycoplasmas. However, the ratio data obtained using the assay methods of the invention showed that 7 days was not sufficient. This was evident from the fact that relationships remained larger than one. Also after treatment removal and continued culture, ratios increased, and cultures were positive again after PCR testing (Stratagene kit). These data are presented in Figure 3, where three different cell lines were observed to be contaminated with M. hyorhinis.

While K562 and U937 cells are suspended cell lines, A549 cells are an adherent cell type; These data therefore confirm that the assay can be used in both adherent and suspended cell types. This is also shown in Figure 2, where CHO and COS-7 cells are adherent cell types commonly used in cell culture laboratories. Figure 3 also shows that 10 day MRA treatment with COS-7 and CHO cell cultures was sufficient to remove contaminating mycoplasmas. EXAMPLE 3: BACTERIAL FILTERS FAILURE TO EXCLUDE MICOPLASMS.

The inventors also showed that culture supernatants passed through various bacterial retardation filters continued to show positive relationships, which is indicative of the presence of viable mycoplasmas. This is shown in Figure 4.

Mycoplasmas can form colonies as large as 600 μτη in diameter, but can also exist in their life cycle as single cells as small as 0.15 μηχ Because of their small size, mycoplasmas can pass through 0.45 µm filters. pm and 0.22 μηι 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 reduced detection sensitivity. Accordingly, it is preferable that the assay methods of the invention be performed on samples that are substantially free of cells. This can easily be achieved by centrifuging cell cultures and sampling the supernatant and optionally by filtration through a bacterial filter. EXAMPLE 4: SENSITIVITY OF PREFERRED TESTS.

As shown in Figures 5 and 8, the supernatant dilution shows the sensitivity of the assays, where a 1: 1000 dilution of contaminated culture supernatant may still give ratios greater than one. Depending on the specific activity of the enzymes acetate and carbamate kinase and in the different mycoplasma species (Mollicutes), it is also possible to separate samples by dilution. The dilution range will also vary according to the number of colony forming units in the test sample. EXAMPLE 5: VARIATIONS OF THE INVENTION TEST METHODS.

The assay methods of the invention are developed without the external exogenous addition of carbamate and acetate kinase substrates in the form of ADP, carbamoyl phosphate and acetyl phosphate or their precursors. In a contaminated culture sample, acetyl or carbamoyl phosphates or their precursors will be endogenously present along with sufficient cell culture derived ADP to initiate the reaction for ATP formation. Alternatively, ADP may be generated by other highly added enzymes or cellular enzymes, i.e. adenylate kinase using ATP and AMP. It is possible to avoid the direct addition of these substrates and have the system generate itself. Using acetate and ammonia together with ATP will cause the acetate kinase and carbamate kinase enzymes to generate acetyl phosphate and carbamoyl phosphate which can then be used by the same enzymes to generate ATP from ADP: The two substrates they may also be generated from "precursors" by utilizing prior parts of glucose fermentation and argenine lysis pathways, for example by the addition of acetyl-CoA and citrulline which can be used by mycoplasmic enzymes to synthesize acetyl phosphate and carbamoyl phosphate, respectively.

The following figures show the differences between the M. fermentam biochemical activities, which generate ATP preferably via the carbamate kinase pathway, but will also use the acetate kinase pathway. Figure 6 shows the effect of adding substrates to the enzyme pathways individually, and then to a combined reagent.

Although M. fermentate uses both pathways, M. orale uses only the carbamate kinase pathway and, as shown in Figure 7, positive relationships are only observed in the individual carbamate reagent or in the combined reagent. Figure 8 shows that detection limits are as low as 14 CFU / reservoir with M orale.

The inventors have researched a mycoplasma which preferably uses the acetate kinase pathway, namely M. hyorhinis. The data are shown in Figure 9.

The inventors tested over 15 different cell lines (see Table 3) and showed that none of the cells have sufficient background enzymatic activity to focus on relationships and give false positives. The inventors, without wishing to be bound by theory, think that the reason for this is that the pathways are anaerobic, and that all mammalian cell cultures will generate ATP through oxidative phosphorylation. Hence, by the use of carbamoyl phosphate alone or a precursor thereof, or acetyl phosphate alone or a precursor thereof, a test method of the invention can be produced which allows the determination of whether the mycoplasmic contaminants in question are of a group using the acetate kinase pathway, the carbamate kinase pathway, or both. This may have useful diagnostic applications.

The only bacteria that have acetate kinase activity are not those that are commonly found as cell culture contaminants, with the possible exception of certain species of E. coli that are manipulated in laboratories, primarily for molecular biology purposes. However, activity with this organism is only observed at very high inoculum concentrations, where there is turbid growth and the resulting turbidity of the sample is easily observed with the naked eye. Accordingly, the methods of the invention may be varied to include an initial screening step for bacterial contamination, if necessary. This can be achieved by a variety of methods, but is preferably accomplished by passing the sample through a standard bacterial filter (Baseman and Tully, 1997).

EXAMPLE 6: PREFERRED REAGENT COMPONENTS FOR USE IN THE MICROPLASM TEST METHODS OF THE INVENTION 1. Mycoplasma Detection Reagent (MDR) per 100 ml • Magnesium acetate 214.5 mg (10 mM) • Inorganic pyrophosphate 178.4 μg (4 μΜ) • Serum albumin 160 mg (0.16%) • D-Luciferin 10 mg (360 μΜ) • L-Luciferine 250 μg (8.9 μΜ) • Luciferase (RY) 85 μg • ADP 250, 5 μg (5 μΜ) • AMP 69.44 mg (2 mM) * RY is the name given to the recombinant luciferase supplied by Lucigen. A mixture of D and L-Luciferin was observed 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 45.87 mg (3 mM) Mycoplasma Substrate (Ms) Precursors Examples of reactions that generate acetyl or carbamoyl: Preferred concentration ranges: 0.1 mM to 100 mM acetyl CoA

Inorganic phosphate 0.1 mM to 100 mM (eg potassium phosphate) Preferred concentration ranges: 100 mM 1 mM citrulline

1 mM to 200 mM ammonium bicarbonate

Preferred concentration ranges: 1 mM acetate (e.g. sodium or potassium) at 500 mM 0.1 mM at 100 mM ATP

Preferred Concentration Ranges: 1mM to 200mM Ammonium Baking

ATP 0.1mMal00mM

Suppliers: Sigma- Aldrich Company Ltd.

Fancy Road Poole Dorset BH12 4QH United Kingdom 3. Mycoplasma Assay Buffer (MAB) per 100 ml • HEPES 1.1915 g (50 mM) • EDTA 74.44 mg (2 mM) • TritonX-100 250 μΐ (0, 25%) • pH 7.50 Preferred concentration ranges of the Components for use in the mycoplasma assay methods and kits of the invention Preferred concentration ranges include • ADP 1 μΜ to 100 mM, preferably 1 to 100 μΜ, more preferably 5 μΜ . • AMP 1 μΜ at 100 mM, preferably 0.1 mM at 10 mM, most preferably 2 mM. Acetyl phosphate 1 μΜ at 100 mM, preferably 0.1 mM to 20 mM, more preferably 3 mM. Concentrations above 10 reduce light output, but the test is still performed. • 1 μΜ to 100 mM carbamoyl phosphate, preferably 0.1 mM to 20 mM, more preferably 3 mM.

EXAMPLE 7: EFFECTS OF DETERGENTS ON THE MICOPLASM TEST The disruption of the viable mycoplasma cell membrane to consider release of enzymes in the sample is a preferred embodiment of the assay method of the invention. This takes into account substrate binding and ATP generation. However, positive relationships indicating mycoplasma contamination can be obtained in the absence of any lysis treatment. The implication is that these enzymes can be released by viable mycoplasma. The other possibility is that some non-viable organisms have released their contents through natural lysis. The addition of even low concentrations of Triton-X100 nonionic detergent greatly increases assay sensitivity by ensuring maximum release of carbamate and acetate kinases in the samples. The purpose of the following experiments was to determine Triton-X100 concentrations in Hepes-EDTA buffer in the relationships observed with mycoplasma contaminated K562 cell cultures. Two organisms were surveyed, M. hyorhinis and M. orale. Figure 10 shows that it is possible to detect mycoplasmatic enzymes in the absence of a detergent lysis step. It also shows a drop in light output at concentrations greater than 4 to 5%, which is due to adverse effects of the detergent on the luciferase enzyme / reaction. However, it is still possible to detect positive activity at concentrations as high as 20% (v / v). The inventors have also shown that the concentrations of Triton-100 used in the above experiments did not result in any detectable carbamate or acetate kinase activity of E. coli strain JM109 cells.

The above results confirm that there is no positive relationship to the number of bacterial cells used. Increasing the concentration of Triton-100 to levels that have been reported to lyse bacterial cells (1-2%) has not yet resulted in positive relationships above.

Generally biological detergents are commonly used to disrupt lipid bipolar membranes to release and then solubilize membrane bound proteins. Nonionic detergents are non-denaturing and allow membrane solubilization without interfering with biological activity. They have been mainly used for the study of protein conformations and for the separation of hydrophilic proteins from membrane-spanning hydrophobic proteins. Anionic and cationic detergents result in greater modification of protein structure and are more effective in disruptive protein aggregation. Zwitterionic detergents are also low in denaturation, but are effective in breaking up protein aggregates.

These different detergent groups have been studied with several different cell types to efficiently lyse and release and preserve the protein content of both eukaryotic and prokaryotic organisms.

As for the preferred assays of the invention, the required lysing agent is one that causes mycoplasmatic membrane disruption and allows the release of metabolic enzymes that are required to react with the substrates. As there is no detergent removal or neutralization step, it is therefore important that the system chosen does not interfere with carbamate and / or acetate kinase activity, or with the luciferase / luciferin / ATP reaction. It is also preferable to use a system that selectively causes mycoplasma lysis, with little or no effect on bacteria that could be potentially contaminating cell cultures / samples. The presence of a filtration step through 0.45 pm filters, however, should remove any larger contaminating microorganisms. The key difference between bacteria and mycoplasmas is the absence of a cell wall, and it is the bacterial cell wall that makes the most difficult bacteria to lyse. There are several very brutal methods that can have total lysis, which include French Press and sonication. Other methods of enzyme digestion include lysozyme followed by the addition of detergents. However, mycoplasma can be lysed with Triton X-100 concentrations around 1 to 2%.

Low concentrations of other nonionic detergents such as Brij®35 (0.4%) (Sigma-Aldrich Company Ltd.) and B-PER (1%) (Perbio Science UK Ltd.) have no adverse effects on the enzyme. luciferase, and are capable of disrupting the mycoplasmic membrane without adversely affecting the luciferase reaction. Concentrations of these detergents can be taken up to 10% without loss of sensitivity of mycoplasmic detection. Contaminating mycoplasma can be detected in the absence of a lysis step to rupture the mycoplasmic membrane. However, the addition of a gentle lysis step (0.25% Triton X-100 in Hepes-EDTA buffer) increases assay sensitivity by releasing the mycoplasmic enzymes of interest into the reaction mixture. The lysis step should preferably cause selective mycoplasma lysis while having little or no effect on bacterial cells. Low concentrations of most nonionic detergents should thus act. However, the filtration step should physically remove any contaminating bacteria, and consider the use of any detergent, but preferably those that do not inhibit either the luciferase reaction or carbamate kinase and acetate kinase activity. EXAMPLE 8: PREFERRED KIT CONTENTS LT07-118 (Sufficient for 10 tests) 1. LT27-217 Mycoplasma Detection Reagent, Lyophilized. 2 x 600 μΐ flasks. 2. LT27-218 Mycoplasma Assay Buffer. 1 x 10 ml bottle. 3. LT27-221 Mycoplasma Substrate. Freeze dried. 2 x 600 μΐ flasks. LT07-218 (Sufficient for 25 tests) 1. LT27-217 Mycoplasma Detection Reagent, Lyophilized. 5 x 600 μΐ flasks. 2. LT27-218 Mycoplasma Assay Buffer. 1 x 10 ml bottle. 3. LT27-221 Mycoplasma Substrate. Freeze dried. 5 x 600 μΐ flasks. LT07-318 (Sufficient for 100 tests) 1. LT27-216 Mycoplasma Detection Reagent, Lyophilized. 1 x 10 ml bottles. 2. LT27-220 Mycoplasma Assay Buffer. 1 x 20 ml bottle. 3. LT27-224 Mycoplasma Substrate. Freeze dried. 1 x 10 ml bottles.

PREFERRED REAGENT COMPOSITIONS FOR KITS AND METHODS OF THE INVENTION 1. Mycoplasma Detection Reagent (MDR) per 100 ml • Magnesium acetate1 214.5 mg (10 mM) • Inorganic pyrophosphate1 178.4 μg (4 μΜ) • Bovine Serum Albumin1 160 mg (0.16%) • D-Luciferin2 10 mg (360 μΜ) • L-Luciferin2 250 μg (8.9 μΜ) • Luciferase (RY) 3 85 μg • ADP1 250.5 μg (5 μΜ) • AMP1 69.44 mg (2 mM) 2. Mycoplasma Substrate (MS) per 100 ml • Acetyl Phosphate 55.23 mg (3 mM) • Carbamoyl Phosphate 45.87 mg (3 mM) 3. Mycoplasma Assay Buffer (MAB) per 100 ml • HEPES 1.1915 g (50 mM) • EDTA 74.44 mg (2 mM) • TritonX-100 250 μΐ (0.25%) • pH 7.50 Preferred concentration ranges: • ADPlpMalOOmM

• AMP 1 pMa 100 mM

• Acetyl phosphate 1 μΜ to 100 mM, preferably mM to 10 mM • Carbamoyl phosphate 1 μΜ to 100 mM Suppliers 1.

Sigma-Aldrich Company Ltd.

Fancy Road Poole Dorset BH12 4QH United Kingdom 2.

Resem BV Goudenregenstraat 84 NL-4131 BE Vanen The Netherlands 3.

Lucigen Ltd.

Porton Down Science Park Porton, Salisbury Wiltshire SP4 OJQ United Kingdom The preferred embodiment of the invention provides a selective biochemical test that takes advantage of the activity of certain mycoplasmic enzymes. The presence of these enzymes provides a rapid screening procedure, enabling sensitive detection of contaminating mycoplasmas in a test sample. Viable mycoplasmas are lysed and the enzymes react with the Mycoplasma Substrate that catalyzes the conversion of ADP to ATP.

By measuring the ATP level in a sample, both before (A) and after (B) the addition of the Mycoplasma Substrate, a BIA ratio that is indicative of the presence or absence of mycoplasma can be obtained. If these enzymes are not present, the second reading will show no increase over the first (A), whereas the reaction of mycoplasmic enzymes with their specific substrates in the Mycoplasma Substrate Reagent leads to high levels of ATP. This increase in ATP can be detected using the following bioluminescent reaction. The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer. The assay is preferably conducted at room temperature (18 to 22 ° C), the optimal temperature for luciferase activity.

SIMPLE TEST PROTOCOL OF THE INVENTION

If j is greater than one = contaminated mycoplasma sample.

If j is one or less = mycoplasma free sample. METHOD OUTLINE It is preferable that the culture supernatant is centrifuged to remove cells and optionally passed through a bacterial filter prior to performing the assay. The kit contains all reagents required to perform the assay. 100 μΐ of the culture supernatant is taken as the sample.

Add Mycoplasma Detection Reagent (MDR) Wait 5 Minutes Read Luminescence (A) Add Mycoplasma Substrate (MS) Wait 10 Minutes.

Read luminescence (B).

Reagent Reconstitution and Storage Make sure that you follow the correct reagent reconstitution applicable to the relevant kit (10.25 or 100 assay points).

This procedure usually requires at least 15 minutes of equilibration time. Mycoplasma Detection Reagent (MDR) and Mycoplasma Substrate (MS) are preferably supplied as lyophilized pellets. These are reconstituted in Mycoplasma Assay Buffer (MAB) to produce working solutions for use in the assay.

For 10 Tests (KIT LT07-118) 1. Preparation of Mycoplasma Detection Reagent Add 600 μΐ of Mycoplasma Assay Buffer to a vial containing the lyophilized Mycoplasma Detection Reagent. Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 2. Preparation of Mycoplasma Substrate Add 600 μΐ of Mycoplasma Assay Buffer to a vial containing lyophilized Mycoplasma Substrate.

Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 3. Mycoplasma Assay Buffer This is preferably supplied ready to use. Store at 2 to 8 ° C when not in use.

For 25 Tests (KIT LT07-218) 1. Preparation of Mycoplasma Detection Reagent Add 600 μΐ of Mycoplasma Assay Buffer to a vial containing the lyophilized Mycoplasma Detection Reagent. Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 2. Preparation of Mycoplasma Substrate Add 600 μΐ of Mycoplasma Assay Buffer to a vial containing lyophilized Mycoplasma Substrate.

Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 3. Mycoplasma Assay Buffer This is preferably supplied ready to use. Store at 2 to 8 ° C when not in use.

For 100 Tests (KIT LT07-318) 1. Preparation of Mycoplasma Detection Reagent Add 10 ml of Mycoplasma Assay Buffer to a vial containing the lyophilized Mycoplasma Detection Reagent.

Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 2. Preparation of Mycoplasma Substrate Add 600 μΐ of Mycoplasma Assay Buffer to a vial containing lyophilized Mycoplasma Substrate.

Replace the lid and mix gently.

Allow the reagent to equilibrate for 15 minutes at room temperature. 3. Mycoplasma Assay Buffer This is preferably supplied ready to use. Store at 2 to 8 ° C when not in use.

Equipment 1. Instrumentation The kit requires the use of a luminometer. The luminometer parameters must be evaluated and the conditions below used to produce the correct machine programming. The preferred assay of the invention was designed for use with cuvette / tube luminometers. For use with plate luminometers, please note below.

Bucket / Tube Luminometers: 1 second reading time (integrated). 2. Additional equipment and consumables a. Sterile 10 ml pipettes b. Luminometer cuvettes c. Micropipettes - 50 to 200 μΐ; 200 to 1000 μΐ d. Countertop centrifuge.

Preferred Test Protocol Please note that culture media samples should be taken prior to any other processing steps, eg trypsinization. 1. Bring all reagents to room temperature before use. 2. Reconstitute Mycoplasma Detection Reagent and Mycoplasma Substrate in Mycoplasma Assay Buffer. Leave for 15 minutes at room temperature to ensure complete rehydration. 3. Transfer 2 ml of the cell culture or culture supernatant to a centrifuge tube and pellet any cells at 1500 rpm (200 x g) for 5 minutes. 4. Transfer 100 μΐ of purified supernatant to the cuvette / tube of a luminometer. 5. Program the luminometer to take 1 second of integrated reading (this is usually the basic parameter on most cuvette luminometers). 6. Add 100 μΐ Mycoplasma Detection Reagent to each sample and wait 5 minutes. 7. Place the cuvette in the luminometer and start the program (Reading A). 8. Add 100 μΐ Mycoplasma Substrate to each sample and wait 10 minutes. 9. Place the cuvette on the luminometer and start the program (Read B). 10. Calculate Ratio = Read B / Read A.

Interpretation of Results The ratio of Read B to Read A is used to determine if a cell culture is contaminated with mycoplasmas. The speed and convenience offered by the kits according to the invention means that it provides a unique method for screening cultures for the presence of mycoplasmas. As such, it is perfectly suited for routine testing of cultured cells. Frequent use of the test methods of the invention will indicate when a cell line becomes infected, enabling prompt remedial action to be taken. The test methods of the invention may also be extended to incoming cell lines and commonly used constituents of the complete media. Interpretation of the different relationships obtained within each experimental situation may vary according to the cell types and conditions used. However, the test gives BI A ratios of less than 1 with uninfected cultures. Cells that are infected with mycoplasma will routinely produce ratios greater than 1. TABLE INTERPRETATION OF TEST RESULTS: ILLUSTRATIVE EXAMPLES OF HEALTHY AND INFECTED CELL LINES__________________________________________________________ Protocol for Plate Luminometers 1. Bring all reagents to room temperature prior to use. 2. Reconstitute Mycoplasma Detection Reagent and Mycoplasma Substrate in Mycoplasma Assay Buffer. Leave for 15 minutes at room temperature to ensure complete rehydration. 3. Transfer 2 ml of cell culture from cell culture supernatant to a centrifuge tube and pellet any cells at 1500 rpm (200 x g) for 5 minutes. 4. Transfer 100 μΐ of purified supernatant to a luminescence compatible plate. 5. Program the luminometer to take an integrated 1 second reading. 6. Add 100 μΐ Mycoplasma Detection Reagent to each sample and wait 5 minutes. 7. Place the plate on the luminometer and start the program (Read A). 8. Add 100 μΐ Mycoplasma Substrate to each sample and wait 10 minutes. 9. Place the plate on the luminometer and start the program (Read B). 10. Calculate Ratio = Read B / Read A.

Great care must be taken when handling any of the reagents. Skin has high levels of ATP on its surface that can contaminate reagents, leading to falsely elevated readings. Latex gloves prevent this problem. The optimum working temperature for all reagents is 22 ° C. If reagents have been refrigerated, always allow them to reach room temperature (18 to 22 ° C) prior to use. The sensitivity of the assay takes into account the detection of hidden contamination and, if the ratio is marginally above 1 (eg up to 1.3), it is recommended that the sample be retested. Any quarantined cultures can be tested after another 24 to 48 hours in culture to see if the ratios have increased.

SUMMARY

Assays of the invention may be performed in the presence or absence of cells. Unlike known mycoplasma detection systems, they take into account samples to be examined quickly using inexpensive portable luminometer systems, and can give the results within 15 minutes to take proper handling of contaminated samples into account. . PCR and DAPI / Hoechst staining will bind to all DNA, whether viable or non-viable mycoplasma. Consequently, if treating and removing mycoplasmas, false positives can still be eliminated when using PCR / DNA staining even though the mycoplasma has been eradicated.

Assays can detect viable mycoplasmas, whereas known methods, such as PCR, cannot distinguish between viable and non-viable mycoplasma.

REFERENCES 1) Razin, S., Yogev, D. and Naot, Y. 1998. Molecular biology and pathogenicity of mycoplasmas. Microbiol. and Mol. Biol Rev. 62 (4): 1094-1156. 2) Rottem, S. and Barile, M.F 1993. Beware of mycoplasmas. TIBTECH. 11: 143-151. 3) Rottem, S. 2002. Sterols and acylated proteins in mycoplasmas. Biochem. Biophys. Res. Commun. 292: 1289-1292. 4) McGarrity, G.J. and Kotani, H. 1985. in The Mycoplasmas Vol. IV. (Razin, S. and Barile, M.F. eds.) Pp. 353-390. Academic Press. 5) Battaglia, M., Pozzi, D., Grimaldi, S. and Parasassi, T. 1994. Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: a method for reducing fluorescence photobleaching. Biotechnic andHistochem. 69: 152-156. 6) Raab, L. S. 1999. Cultural revolution: mycoplasma testing kits and Services. The Scientist 13 (20): 21-25. 7) Verhoef, V., Germain, G. and Fridland, A. 1983. Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells. Exp. Cell Res. 149 (1): 37-44. 8) Whitaker, A.M., Windsor, G.D., Bumett, C.M. and Taylor, C.H. 1987. A rapid and sensitive method for detection of mycoplasmas in infected cell cultures using 6-methyl purine deoxyriboside. Dev. Biol. Stand 66: 503-509. 9) Daxboeck, F., Krause, R. and Wenisch, C. 2003. Laboratory diagnosis of Mycoplasma pneumoniae infection. Clin. Microbiol. Infect 9 (4): 263-273. 10) from Wet, J. R., Wood, K. V., De Luca, M., Helinski, D. R. and Subramani, S. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell Biol. 7 (2): 725-737. 11) Masuda, T., Tatsumi, H. and Nakano, E. 1989. Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata. Gene. 77 (2): 265-270. 12) Wood, K. V., Lam, Y. A., Seliger, Η. H. and McElroy, W. D. 1989. Complementary DNA coding click beetle luciferase can elicit bioluminescence of different colors. Science 244 (4905): 700-702. 13) Wet, J.R., Wood, K.V., Helinski, D.R. and DeLuca, M. 1986. Cloning firefly luciferase. Methods Enzymol. 133: 3-14. 14) White, P.J., Squirrell, D.J., Amaud, P., Lowe, C.R. and Murray, J.A. 1996. Improved thermosiability of the North American firefly luciferase: saturation mutagenesis at position 354. Biochem. J. 319 (2): 343-350. 15) Baseman, J. B. and Tully, J. G. 1997. Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerg. Infect Dis. 3 (1): 21-32. 16) Kirchoff, H., Mohan, K., Schmidt, R., Runge, M., Brown, D. R., Brown, Μ. B., Foggin, C. M., Muvavarirwa, P., Lehmann, H. and Flossdorf, J. 1997. Mycoplasma crocodyli sp. nov., a mew species from crocodiles. Int. J. Syst. Bacieriol. 47: 742-746. 17) Forsyth, Μ. H., Tully, J. G., Gorton, T. S., Hinkley, L., Frasca, S., van Kruiningen, H. J. and Geary, S. J. Int. J. Syst. Bacteriol. 1996. Mycoplasma sturni sp. nov., from the conjunctiva of a European starling (Stumus vulgaris). Int. J. Syst. Bacteriol. 46: 716-719. 18) Taylor, R.R., Mohan, K. and Miles, R.J. 1996. Diversity of energy-yielding substrates and metabolism in avian mycoplasmas. Vet. Microbiol. 51: 291-304. 19) Tully, J.G., Whitcomb, R.F., Rose, D.L., Bove, J.M., Carie, P., Somerson, N.L., Williamson, D.L., and Eden-Green, S. 1994. Acholeplasma brassica sp. Nov and Acholeplasma palmae sp. nov., two non-sterol-requinng mollicutes from plant surfaces. Int. J. Syst. Bacteriol. 44: 690-694. 20) Web Reference: www.unc.edu/depts/tcf/mvcoplasma.htm 21) Duffy, LB, Crabb, D., Searcey, K. and Kempf, MC 2000. Comparative potency of gemigloxacin, new quinolones, macrolides, teiracycline and clindamycin against Mycoplasma spp. J. Antimicrobial Chemotherapy. 45: 29. 22) Taylor-Robinson, D. and Bebear, C. 1997. Antibiotic susceptibilities of mycoplasmas and treatment of mycoplasmal infections. 40: 622-630. 23) Uphoff, C.C., Meyer, C. and Drexler, H.G. 2002. Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics. 16 (2): 284-288. 24) Schram, E. and Weyens-van Witzenburg, A. 1989. Improved ATP methodology for biomass assays. J. Biolumin. Chemilumin. 4: 390-398. 25) Stanley, P. E. 1989. A review of bioluminescent ATP techniques in rapid microbiology. J. Biolumin. Chemilumin. 4: 375-380. 26) Pellegrini, A., Thomas, U., von Fellenberg, R. and Wild, P. 1992. Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character. Appl Bacteriol. 72: 180-187.

Claims (34)

1. A method for detecting the presence of mycobacteria in a test sample, said mycoplasma being a contaminant mycobacteria characterized by the fact that it comprises; (i) provide a test sample; (ii) detecting and / or measuring acetate kinase and / or carbamate kinase activity (B) in the test sample and said activity indicating the presence of contaminating mycoplasma; and (iii) identifying the test sample when contaminated with mycoplasma based on detecting and / or measuring said activity from step (ii). Method according to claim 1, characterized in that it further comprises the following steps performed after step (ii) but before step (iii): (iia) obtaining information (A) of acetate kinase activity and / or carbamate kinase detected and / or measured in a corresponding control sample; and (iib) compare the activity detected and / or measured in the test sample (B) with that of the control sample (A); wherein the test sample is identified as contaminated with mycoplasma in step (iii) if activity (B) detected and / or measured in the test sample in step (ii) is greater than that of the control sample (A) in step (lia), that is, the ratio BfA is greater than one, Method according to claim 1 or 2, characterized in that it detects and / or measures the activity (B) of acetate kinase and / or carbamate kinase in the test sample in step (ii) and / or obtains the Information (A) on acetate kinase and / or carbamate kinase activity in a corresponding control sample in step (iia) comprises detecting and / or measuring the appearance and / or disappearance of one or more of the substrates and / or one or more. products of the following reactions: Method according to any one of claims 1 to 3, characterized in that it further comprises the step of releasing the cellular contents of mycoplasmas into the sample by treating the test sample with a mycoplasma lysis agent which is performed after step (i), but before step (ii). A method according to claim 4, characterized in that the lysing agent is a detergent, Method according to claim 5, characterized in that the detergent lysing treatment is not able to lyse bacterial cells. A method according to any one of claims 4 to 6, characterized in that the corresponding control sample is the same test sample prior to mycoplasma lyase treatment. A method according to any one of claims 2 to 4, characterized in that the corresponding control sample is the same test sample, but the detection / measurement for test sample activity information is obtained. after a time interval after obtaining the detection / measurement information for the control sample. Method according to claim 8, characterized in that the time interval is 30 minutes. Method according to any one of claims 1 to 9, characterized in that the step of detecting and / or measuring comprises detecting and / or measuring ATP. Method according to Claim 10, characterized in that the ATP is detected and / or measured by a light-emitting reaction. Method according to Claim 11, characterized in that the light-emitting reaction is a bioluminescent reaction. Method according to any one of the preceding claims, characterized in that the ADP is added to the test sample prior to step (ii) of detecting and / or measuring. Method according to any one of the preceding claims, characterized in that a mycoplasma substrate (MS) reagent is added to the test sample prior to step (ii) of detecting and / or measuring, the MS reagent comprising : acetyl phosphate or acetyl CoA and / or carbamoyl or citrulline phosphate and / or ammonia. Method according to claim 13 or 14, characterized in that the control sample is all or an aliquot of the test sample to which a mycoplasma reagent has not been added. A method according to any one of claims 2 to 7, or 9 to 15, characterized in that the control sample was presented as free of mycoplasma by a separate method. A method according to claim 16, characterized in that the control sample was presented as free of mycoplasma by one or more of the PCR tests, DNA fluorescence staining, or mycoplasma culture method. A method according to any preceding claim, characterized in that the test sample and / or control sample is a cell culture sample. Method according to claim 18, characterized in that the cells in the cell culture sample are mammalian cells, preferably adherent cells, such as Vero, MRC5, HUVEC, BSMC, NHEK, MCF-7, AoSMC cells. , A549, HepG2, FM3A, PC 12, ARPE-19, CHO and COS, and / or adherent primary cell isolated from animal source. Method according to claim 18, characterized in that the mammalian cells in the cell culture sample are grown in suspension, such as K562, U937, HL-60, Cem-7, and Jurkats plus primary cell types such as like the blast cells of leukemia. Method according to claim 18, characterized in that the cell culture is a plant cell culture. A method according to any one of claims 18 to 21, characterized in that the cell culture sample is a sample that is derived from a cell culture but is itself substantially free of cell material. Method according to any one of claims 1 to 17, characterized in that the test sample and / or control sample consist of a cell-free reagent. The method according to claim 23, characterized in that the cell free reagent is trypsin. Method for detecting the presence of mycoplasma in a test sample, characterized in that it comprises the following steps: (i) providing a test sample; (ii) without adding an exogenous reagent (eg substrates for kinase activity) transforming ADP into ATP, detecting or measuring ATP in the test sample using a bioluminescent reaction to obtain ATP and / or light output measurement (A ); (iii) obtain an ATP and / or measure light output (B) from a corresponding control sample; (iv) compare the ratio of ATP and / or B / A light output measurement; and (v) identify the test sample as contaminated with mycoplasma in the event that the BI A ratio is greater than one. Method according to any one of the preceding claims, characterized in that it includes a step of passing the test sample through a filter that holds the bacterial cells. Use of acetyl phosphate or a precursor thereof and / or carbamoyl phosphate or a precursor thereof, characterized in that it is in a method of detecting mycoplasma contamination of a sample as defined in claims 14 to 26. 28. Kit for use in detecting mycoplasma contamination, characterized in that it comprises the following: (i) acetyl phosphate or acetyl CoA and / or carbamoyl phosphate or citrulline and / or ammonia; (ii) ADP in an excess amount to drive enzymatic reactions toward ATP formation; (iii) one or more agents for lysing mycoplasma; (iv) means for detecting and / or measuring ATP by a light emitting reaction; (v) a mycoplasma assay buffer (MAB) into which lyophilized reagents may be reconstituted. A kit according to claim 28, characterized in that the mycoplasma lysing agents comprise a detergent. A kit according to claim 28, characterized in that said means comprise a mycoplasma detection reagent (MDR) comprising magnesium acetate, inorganic pyrophosphate, bovine serum albumin, luciferin, luciferase, ADP and AMP. A kit according to any one of claims 28 to 30, characterized in that the reagents are provided in a lyophilized condition. Kit according to claim 28, characterized in that the buffer maintains a pH of approximately 7.5. A kit according to claim 28 or 30, further comprising a luminometer, which is preferably a portable luminometer. Kit according to any one of claims 28 to 33, characterized in that it further comprises a bacterial filter.