# Patent

## Title
Mycoplasma detection method and composition

## Abstract

A method of detecting the presence of mycoplasma in a sample and a mycoplasm detection kit. A sample is contacted with a first and a second oligonucleotide probe, wherein the first and second oligonucleotide probes are substantially complementary to portions of a 16S ribosomal subunit of at least one mycoplasma species. The first probe may be labeled with a capture ligand. The second probe may be labeled with a first component of a detection system.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. application Ser. No. 60/716,326, filed Sep. 12, 2005, the disclosure of which is incorporated herein by reference.


FIELD OF THE INVENTION
This invention relates to the methods and kits for the detection of Mycoplasma contamination in cell cultures or other samples.
BACKGROUND OF THE INVENTION
Mycoplasma is a term used to denote a species included in the class Mollicutes. They are the smallest and simplest free-living parasitic organisms known. Mycoplasma are parasites of many animal species and typically exhibit host and tissue specificity. In humans, Mycoplasma (M.) pneumoniae is the respiratory pathogen responsible for atypical pneumonia. Mycoplasma are frequently isolated from patients with immunodeficiencies associated with disease states.
Mycoplasma are common contaminants of eukaryotic cell cultures and are known to alter the phenotypic characteristics of host cell lines. The published incidence of mycoplasma infected cell cultures has ranged from 4 to 92%. Of the 18 most common species recognized as culture contaminants, M. orale, M. hyorhinis, M. arginini, M. fermentans, and Acholeplasma (A.) laidlawii are the most frequently isolated representing 80 to 90% of all isolates. The small size of mycoplasma allows them to pass through the commonly used 0.45 μm sterilization filters and mycoplasma are typically resistant to antibiotics such as penicillin and streptomycin. Mycoplasma contamination usually does not produce visible changes in cell culture medium despite the fact it can reach titers of 108 per milliliter. Sources of mycoplasma contamination include laboratory personnel, reagents, and mycoplasma contaminated cell lines.
Mycoplasma contamination is detected by a number of methods. Microbial culture is generally considered the most sensitive method for mycoplasma screening and is commonly used as a reference for the evaluation of any new mycoplasma detection techniques. However, culturing mycoplasma can take two to four weeks, and cannot detect fastidious mycoplasma. Moreover, culturing mycoplasma requires special growth conditions and is generally restricted to specialized laboratories.
Another method involves microscopic visualization of mycoplasma attached to host cells using fluorescent DNA staining that displays extra-cellular fluorescence as well as extra-nuclear fluorescence. This method is efficient for mycoplasma screening and particularly for detection of M. hyorhinis strains that cannot be cultivated on microbiological media. However, fluorescent staining cannot detect plasma species that cyto-absorb poorly. Moreover, this method requires expertise for accurate interpretation of results.
Enzyme-linked immunosorbent assays measuring mycoplasma specific cell-surface antigens have been described but typically lack sensitivity. A number of polymerase chain reaction (PCR) based methods have also been described for the detection of mycoplasma that achieve high sensitivity and may be amenable to species identification. However, PCR-based detection of mycoplasma is prone to false positives due to amplicon contamination and false negative results due to use of excess sample. Mycoplasma screening methods utilizing select biochemical activity have also been used, but give inconsistent results when comparing different cell lines.
While these known methods have provided researchers with various techniques to determine mycoplasma contamination, these techniques have several disadvantages as described above. More efficient, rapid and sensitive methods of detecting contamination are desired.
SUMMARY OF THE INVENTION
The present invention comprises an assay method and kit designed for routine screening of mycoplasma contamination of cultured cells and other samples. In one embodiment, the method and kit of the invention detect mycoplasma 16S ribosomal RNA present in a sample using a calorimetric amplification system with a sensitivity comparable to PCR. In one aspect of the invention, the method of the invention is used to detect the presence of at least one of the most common mycoplasma species found as contaminants in samples such as cell cultures. Those species include M. hyorhinus, M. arginini, M. fermentans, M. orale, M. pirum, M. hominis, M. salivarium, and A. laidlawii. These species account for approximately 95% of all mycoplasma contaminations.
In one embodiment, the invention comprises a method for detecting the presence of mycoplasma in a sample comprising contacting a sample treated to release ribosomal RNA of mycoplasma present in the sample with a first and second oligonucleotide probe wherein the first oligonucleotide probe is substantially complementary to a portion of a 16S ribosomal RNA of at least one mycoplasma species and is labeled with a capture ligand and wherein the second oligonucleotide probe is substantially complementary to a different portion of the 16S ribosomal RNA of the mycoplasma species; incubating the sample with the first and second oligonucleotide probes under hybridization conditions to form a hybridization solution and for a time sufficient for the probes to hybridize to 16S ribosomal RNA of mycoplasma species present in the sample; and contacting the hybridization solution with a solid phase coated with a capture receptor capable of specifically binding to the capture ligand of the first labeled oligonucleotide probe and detecting the presence of mycoplasma ribosomal RNA in the sample.
In one embodiment, the first oligonucleotide probe is one of the following capture probes: SEQ ID NO. 12—ggataacgct tgcaacctat gtattaccg; SEQ ID NO. 13—ggtgtgtaca agacccgaga acgtattcac; SEQ ID NO. 14—ggtgtgtaca aaacccgaga acgtattcac; and SEQ ID NO. 15—ggtgtgtaca aaccccgaga acgtattcac and the second oligonucleotide probe is one of the following probes: SEQ ID NO. 1—atatctacgc attccaccgc ttcacaagg; SEQ ID NO. 2—atatttacgc attttaccgc tacacatgg; SEQ ID NO. 3—gccccactcg taagaggcat gatgatttg; SEQ ID NO. 4—gccctagaca taaggggcat gatgatttg; SEQ ID NO. 5—cgaattgcag acttcaatcc gaactgaga; SEQ ID NO. 6—cgaattgcag actccaatcc gaactgaga; SEQ ID NO. 7—cgagttgcag actacaatcc gaactgaga; SEQ ID NO. 8—cgaattgcag ccctcaatcc gaactgaga; SEQ ID NO. 9—tactactcag gcggatcatt taatgcgtta g; SEQ ID NO. 10—tactactcag gcggagaact taatgcgtta t; and SEQ ID NO. 11—tactacccag gcgggatgtt taatgcgtta g.
In one aspect of the invention, cell culture supernates or cultured cell pellet samples are lysed to form samples. Samples are hybridized with a hybridization solution containing the oligonucleotide probes having the sequences SEQ ID NOs. 12-15 labeled with a capture ligand and oligonucleotide probes having the sequences SEQ ID NOs. 1-11 labeled with a component of a detection system comprising a detection ligand and wherein the probes are targeted to the 16S ribosomal RNA of at least eight of the most common mycoplasma species typically found to contaminate cell cultures, that is: M. hyorhinus, M. arginini, M. fermentans, M. orale, M. pirum, M. hominis, M. salivarium, and A. laidlawii. In one embodiment the capture ligand is biotin and the detectable ligand is digoxigenin. In one method of the invention the hybridization solution is contacted with a solid phase coated with a capture receptor chosen to specifically bind to the capture ligand and the small rRNA probe hybrid is captured. Following a wash to remove unbound material, another component of the detection system comprising a detection receptor chosen to specifically bind to the detection ligand where the detection receptor is bound to a signal generating label, is contacted with the solid phase for a time sufficient for the detection ligand to bind to the detection receptor. A substrate solution that reacts with the signal generating label to produce a detectable signal is then added and the detectable signal is produced in proportion to the presence of mycoplasma rRNA in the original sample. In one embodiment of the invention the signal generating label produces a color, which color development may optionally be amplified by adding an amplifier solution.


DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a method and kit to detect mycoplasma which has contaminated cell cultures in a laboratory setting. The method of the invention includes the use of a first and a second oligonucleotide probe, each being complementary or substantially complementary to a portion of a 16S ribosomal RNA of a mycoplama species. The terms “complementary” and “substantially complementary,” as used herein, refer to sequences that may base pair hybridize. Complementary nucleotides are, generally, adenine (a) and thymine (t) or uracil (u), and cytosine (c) and guanine (g). Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least 50% of the nucleotides of the other strand, preferably at least 60% of the nucleotides, more preferably at least 70% of the nucleotides, still more preferably, at least 80% of the nucleotides, and still more preferably between 90% and 100% of the nucleotides. In one embodiment of this invention, two or more different first oligonucleotide probes are contacted with the sample and two or more different second oligonucleotide probes are contacted with the sample. In this embodiment, each oligonucleotide probe will be substantially complementary to a portion of the 16S ribosomal RNA of a mycoplasma species. In the method of the invention, one second oligonucleotide probe may be substantially complementary with approximately 90% of the nucleotides of the probe pairing with the RNA of one species of mycoplasma present in the sample but may only be substantially complementary with approximately 75% of the nucleotides of the probe pairing with the RNA of another species of mycoplasma present in the sample. However, the presence of both mycoplasma species will be detected using the method of the assay. In one aspect of the invention, each of the first oligonucleotide probes will be substantially complementary with greater than 90% of the nucleotides of the probe pairing with a portion of the 16S ribosomal RNA of each of the eight most common mycoplasma species found to contaminate cultures.
In some embodiments, a first oligonucleotide probe is labeled with a capture ligand that is chosen to bind specifically with a capture receptor bound to a solid phase where the label does not prohibit the hybridization of the first oligonucleotide probe to a portion of the 16S ribosomal RNA of a mycoplasma species. As used herein, the terms “ligand” and “receptor” are used to refer to a reagent or substance that is a binding pair member that is capable of recognizing the specific spatial and/or charge configuration of the other binding pair member and of binding specifically with it, where a ligand is one member of the binding pair and a receptor is the other member of the binding pair.
Binding pairs are well known and include the following: antigen-antibody and nucleic acid-nucleic acid binding protein, biotin and avidin, biotin and streptavidin, carbohydrates and lectins, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, polymeric acids and bases, and the like. Where the binding pair member or reagent is described herein as an antibody, the term “antibody” is intended to encompass an effective portion thereof retaining specific binding activity for the substance or element. Effective portions include, for example, Fv, scFv, Fab, Fab2, and heavy chain variable regions or a chimeric molecule or recombinant molecule or an engineered protein comprising any of the above-mentioned portions.
In some embodiments of the invention, the capture ligand is biotin and the capture receptor is streptavidin, avidin or an anti-biotin antibody.
As used herein, “bound to” or “coated with” with reference to the solid phase encompasses all mechanisms for binding antibodies and proteins and other substances, directly or indirectly to surfaces of solid phases so that when the solid phase is contacted with a solution containing the sample the capture receptor bound to the solid phase remains associated with the surface. Such mechanisms include chemical or biochemical linkage via covalent attachment, attachment via specific biological binding (e.g., biotin/streptavidin), coordinative bonding such as chelate/metal binding, or the like. In one aspect of the invention, streptavidin is linked to the solid surface with chemical attachment.
“Solid phase” as used herein refers to an insoluble material to which one component of the detection method may be bound. Known materials of this type include hydrocarbon polymers such as polystyrene and polypropylene, glass, metals and gels. Such supports may be in the form of beads, tubes, strips, disks, microplates and the like. Polystyrene microplates are desirably used with the detection method of the system.
As used herein, “specific binding” and “specifically bound” means that the reagent, substance or moiety is a binding pair member that binds or is bound to a desired substance or element with a higher binding affinity and/or specificity to the substance or element than to any other moiety present in the sample or used in the assay method.
In some embodiments, a second oligonucleotide probe used is labeled with a first component of a detection system comprising a detection ligand, that does not prevent the hybridization of the second oligonucleotide probe to a portion of the 16S ribosomal RNA of at least one species of mycoplasma. The first component of the detection system will react with a second component of the detection system comprising a detection receptor chosen to specifically bind to the detection ligand and wherein the detection receptor is labeled with a signal generating moiety. The signal generating moiety may be a chemical label such as an enzyme, a fluorescent compound, a radioisotope, a chromophore, or any other signal generating moiety, provided that when the signal generating moiety is bound to the detection receptor the detection receptor retains its capacity to specifically bind to the detection ligand. “Detection system,” and the like, as exemplified below, refers to a chemical system that generates a detectable signal. In one aspect of the invention, the detection system includes as a detection ligand a hapten or protein that may be attached to the second oligonucleotide probe without interfering with its capacity to hybridize with a substantially complementary sequence, including without limitation, digoxigenin. The detection receptor in this embodiment is an antibody to digoxigenin labeled with a signal generating moiety that is an enzyme where the detectable signal may be generated by exposing the labeled reagent to a particular substrate and incubating for a signal such as color, fluorescence or luminescence development. In a preferred embodiment, the enzyme is alkaline phosphatase and the detection systems described in U.S. Pat. Nos. 4,446,231, 4,595,655, and 4,598,042, the relevant portions of which are herein incorporated by reference, are used. Briefly, the detection systems described in those patents discloses a method where the signal generating moiety is an enzyme which converts a precursor in a substrate into a cycling vector which in turn is interconverted in a cycling detection system by contacting it with a secondary system and the signal generated by the enzyme is amplified by the enzyme constantly increasing the amount of cycling factors in the system.
“Kit” as used herein refers to a combination of reagents usually formulated with necessary buffers, salts and stabilizers, where the reagents are premeasured so as to at least substantially optimize the performance of the detection method.
One aspect of the invention provides for the use of a test kit to detect the presence of mycoplasma in a sample. In this embodiment, the kit includes at least two different first oligonucleotide probes. In one embodiment, a mycoplasma detection kit for detecting the presence of mycoplasma contamination comprises two or more different first oligonucleotide probes wherein each first oligonucleotide probe is substantially complementary to a portion of a 16S ribosomal RNA of a mycoplasma species and is labeled with a capture ligand; two or more different second oligonucleotide probes wherein the second oligonucleotide probe is substantially complementary to a different portion of the 16S ribosomal RNA of the mycoplasma species than any of the first oligonucleotide probes and each is labeled with a first component of a detection system, wherein the first component comprises a detection ligand; a solid phase coated with a capture receptor chosen to specifically bind to the capture ligand on the first probe; and a detection solution comprising a second component of the detection system, wherein the second component comprises a detection receptor labeled with a signal generating moiety. In a preferred embodiment, the kit comprises first oligonucleotide probes of each of SEQ ID NOs. 12-15 and second oligonucleotide probes of each of SEQ ID NOs. 1-11.
Embodiments of the invention select the first and second oligonucleotide probes to minimize cross-reactivity with other bacteria species. Embodiments of the invention also select the first and second oligonucleotide probes to minimize the formation of heterodimers of the first and second oligonucleotide probes which can result in high levels of background noise. Thus the choice of appropriately matched first and second oligonucleotide probes is an important aspect of the invention. Embodiments of the invention comprising one or more first oligonucleotide probes selected from ID SEQ NOs. 12-15 and one or more second oligonucleotide probes selected from ID SEQ NOs. 1-11 provide for appropriate matches of first and second oligonucleotide probes for detection of mycoplasm contamination by minimizing crossreactivity with other bacteria species while also minimizing interaction between the probes.
In another embodiment of the invention, the signal generating moiety is an enzyme and the kit further includes a substrate solution that includes reagents that will react with the enzyme to generate a detectable signal, and in a further aspect of the invention the kit further includes an amplifier solution comprising reagents that will amplify the generation of the detectable signal being produced.
The kit of the invention may further include a cell lysis diluent that includes water treated with diethylpyrocarbonate (DEPC) at a concentration sufficient to inactivate RNase, RNase-free Trizma hydrochloride (Tris[hydroxymethyl]aminomethane hydrochloride), Trizma base (Tris[hydroxymethyl]aminomethane), calcium chloride dihydrate, and proteinase K.
The following examples are illustrative of the invention and is not intended to limit the scope of the invention as set out in the appended claims.
EXAMPLE 1
The following Example may be performed using the Mycoplasma Detection Kit, the MycoProbe® Mycoplasma Detection Kit, commercially available from R&D Systems, Inc., Minneapolis, Minn.
Sample Preparation
Cell lysate supernates (15 μL/well) or cell pellets (4,700 to 19,000 cells/well) are used as samples in this method. Cell pellet samples are stored on ice until lysed or stored at ≦−20° C. for use at a later time. Cell lysate samples are prepared using the following procedure: Four hundred microliters of cell lysis diluent (diluent included water treated with diethylpyrocarbonate (DEPC) at a concentration sufficient to inactivate RNase, RNase-free Trizma hydrochloride (Tris[hydroxymethyl]aminomethane hydrochloride), Trizma base (Tris[hydroxymethyl]aminomethane), calcium chloride dihydrate, and proteinase K, hereinafter referred to as “Cell Lysis Diluent”) is added to a cell pellet containing 5×105 cells to obtain a final concentration of 1.25×106 cells per milliliter. The cells are pipetted up and down several times until resuspended and vortexed for 15 to 20 seconds. The cell lysate is again diluted with Cell Lysis Diluent to obtain a final concentration of approximately 3×104 to approximately 1.2×105 cells per milliliter.
Cell culture supemate samples are similarly prepared. All cell culture supernate samples are diluted 10-fold in Cell Lysis Diluent and vortexed for 15-20 seconds.
Assay Procedure
The hybridization plate (a 96 well plate) is prepared by washing twice with wash buffer. Excess wash buffer is removed by decanting or aspirating. The plate is inverted and blotted against clean paper towels.
Each well receives 50 microliters of cDNA Probes including all of the oligonucleotide probes listed in Table 1. These probes of Table 1 are the first and second oligonucleotide probes of the invention, wherein each of the first oligonucleotide probes is labeled with biotin and each of the second oligonucleotide probes is labeled with the first component of the detection system, digoxigenin.







TABLE 1







Detection and Capture Probes














SEQ ID
Capture/


Tm
ε(nmoles/
ε(μg/



NO.
Detection
Sequence
Length
(2 + 4)
A260)
A260)

















No. 1
Detection
atatctacgc attccaccgc
29
79.8
3.66
32.4





ttcacaagg


 


No. 2
Detection
atatttacgc attttaccgc
29
74.8
3.60
32.1




tacacatgg


 


No. 3
Detection
gccccactcg taagaggcat
29
81.7
3.58
32.2




gatgatttg


 


No. 4
Detection
gccctagaca taaggggcat
29
79.3
3.47
31.5




gatgatttg


 


No. 5
Detection
cgaattgcag acttcaatcc
29
79.2
3.48
31.3




gaactgaga


 


No. 6
Detection
cgaattgcag actccaatcc
29
80.8
3.50
31.3




gaactgaga


 


No. 7
Detection
cgagttgcag actacaatcc
29
77.6
3.44
31.0




gaactgaga


 


No. 8
Detection
cgaattgcag ccctcaatcc
29
83.9
3.57
31.9




gaactgaga


 


No. 9
Detection
tactactcag gcggatcatt
31
77.0
3.32
31.9




taatgcgtta g


 


No. 10
Detection
tactactcag gcqqagaact
31
76.2
3.29
31.7




taatgcgtta t


 


No. 11
Detection
tactacocag gcgggatgtt
31
81.2
3.33
32.1




taatgcgtta g


 


No. 12
Capture
ggataacgct tgcaacctat
29
76.2
3.57
32.0




gtattaccg


 


No. 13
Capture
ggtgtgtaca agacccgaga
30
77.7
3.34
31.1




acgtattcac


 


No. 14
Capture
ggtgtgtaca aaacccgaga
30
77.6
3.33
31.0




acgtattcac


 


No. 15
Capture
ggtgtgtaca aaccccgaga
30
79.2
3.39
31.4




acgtattcac









One hundred fifty microliters of a positive control, negative control or sample, either the cell lysate or cell culture supemate lysate, are added to each of the designated wells in the prepared hybridization plate and then covered with a plate sealer. The positive control used in this example is complementary to one detection probe and one capture probe. It is intended to provide a strong positive signal to indicate that the assay is performed correctly. The sequence for the positive control for use in this example is SEQ ID NO. 16 caaatcatca tgcctcttac gagtggggcg tgaatacgtt ctcgggtctt gtacacacc. The negative control contains only Cell Lysis Diluent and no cellular sample. A float collar is applied to the hybridization plate and the plate incubated for 60 minutes in an approximately 65 degree C. water bath. A polystyrene microplate coated with streptavidin (the “Streptavidin Plate”) is washed twice with wash buffer and the excess buffer is removed. One hundred fifty microliters of each well of the hybridization plate are then transferred from each well of the hybridization plate to a designated well of the Streptavidin Plate and a new plate sealer is applied. The plate is incubated for 60 minutes at room temperature on a horizontal orbital shaker set at 500±50 rpms. The Streptavidin Plate is washed four times with wash buffer and then excess wash buffer is removed. Each well receives 200 μL of anti-digoxigenin conjugate (21 mL of a polyclonal antibody against digoxigenin, conjugated to alkaline phosphatase) and the plate is covered with a new plate sealer. The plate is again incubated for 60 minutes on the shaker at a temperature of approximately 20-25° C. The Streptavidin Plate is washed six times with wash buffer and then excess wash buffer is removed. 50 μL of substrate solution (lyophilized NADPH with stabilizers in a buffered solution) is added to each well and then the plate is covered with a new plate sealer. The plate is again incubated for 60 minutes on the shaker at a temperature of approximately 20-25° C. The plate is not washed. 50 μL of amplifier solution (buffered solution containing INT-violet with stabilizers) is added to each well and the plate covered with a new plate sealer. The samples are incubated for 30 minutes on the shaker at a temperature of approximately 20-25° C. and not washed. At the end of this time, 50 μL of a stop solution (2 N sulfuric acid) is added to each well. The optical density (OD) of each well was determined within 30 minutes, using a microplate reader set to 490 nm. If wavelength correction is available, the optical density is set to 650 nm or 690 nm. If wavelength correction is not available, readings at 650 nm or 690 nm are subtracted from the readings at 490 nm to correct for optical imperfections in the plate. Readings made directly at 490 nm without correction may be higher and, therefore, less accurate.
The results are calculated by determining the average of the duplicate optical density readings for each control and sample. The average negative control optical density value is subtracted from all average optical density values. The calculated positive control optical density value should be greater than or equal to 1.5. The results from a calculated sample of OD values are determined by using Table 2, below. Through development and validation, it was discovered that values less than 0.05 OD are negative. Samples with values above 0.10 are positive. Values of 0.05-0.10 OD require retesting after a few days. If after a few days the sample continues to have the same OD level (0.05-0.10 OD), then it is negative. However, if the OD level increases, then it is positive.







TABLE 2







Calculation of Results









OD Values




(calculated)
Result
Interpretation





<0.05
Negative
No mycoplasma detected.


0.05-0.10
Inconclusive
Sample is suspect for Mycoplasma. Continue




to culture for an additional 2-3 days and




repeat the test. If sample give a similar




OD, then no Mycoplasma are detected.


>0.10
Positive
Mycoplasma detected.









EXAMPLE 2
The sensitivity of the method of the invention was shown by growing each Mycoplasma species in a pure culture serially diluted and tested with the methods of Example 1. The sensitivity results are set forth in Table 3, below.







TABLE 3







Sensitivity











Sensitivity



Mycoplasma Species
(CFU/well)













Mycoplasma arginini
15



Mycoplasma orale
65



Mycoplasma fermentans
75



Acholeplasma laidlawii
240



Mycoplasma hyorhinis
560



Mycoplasma pirum
30



Mycoplasma hominis
225



Mycoplasma salivarium
2500





CFU = Colony Forming Units






EXAMPLE 3
Sample Preparation
Supemate samples of log phase growth CTLL-2 cells, a mouse cytotoxic T lymphocyte cell line, were tested for possible mycoplasma contamination. Supemates can be harvested at any time and stored on ice or frozen at ≦−20° C. until use. When ready to assay, dilute the supemate sample 10-fold with cell lysis diluent (diluent included water treated with diethylpyrocarbonate (DEPC) at a concentration sufficient to inactivate RNase, RNase-free Trizma hydrochloride (Tris[hydroxymethyl]aminomethane hydrochloride), Trizma base (Tris[hydroxymethyl]aminomethane), calcium chloride dihydrate, and proteinase K, hereinafter referred to as “Cell Lysis Diluent”). Allow the supemate sample to thaw on ice if the sample has been stored at ≦−20° C. For this experiment, 33 μL of supernate sample was diluted with 297 μL of Cell Lysis Diluent, vortexed and stored on ice. The sample can also be frozen at ≦−20° C, until used. If stored at ≦−20° C., sample should be thawed on ice before use.
Assay Procedure
The hybridization plate (a 96 well plate) was prepared by washing twice with wash buffer. Excess wash buffer was removed by decanting or aspirating. The plate was inverted and blotted against clean paper towels.
Each well received 50 microliters of cDNA Probes including all of the oligonucleotide probes listed in Table 1. The probes of Table 1 are the first and second oligonucleotide probes of the invention, wherein each of the first oligonucleotide probes was labeled with biotin and each of the second oligonucleotide probes was labeled with the first component of the detection system, digoxigenin.
One hundred fifty microliters of a positive control, negative control or sample were added to each designated wells in the prepared hybridization plate and then covered with a plate sealer. The positive control used in this example is complementary to one detection probe and one capture probe. It was intended to provide a strong positive signal to indicate that the assay was performed correctly. The sequence for the positive control used in this example was ID SEQ NO. 16 caaatcatca tgcctcttac gagtggggcg tgaatacgtt ctcgggtctt gtacacacc. The negative control contained only Cell Lysis Diluent buffer and no cellular sample. A float collar was applied to the hybridization plate and the plate incubated for 60 minutes in an approximately 65 degree C. water bath. A polystyrene microplate coated with streptavidin (the “Streptavidin Plate”) was washed twice with wash buffer and the excess buffer was removed. One hundred fifty microliters of each well of the hybridization plate were then transferred from each well of the hybridization plate to a designated well of the Streptavidin Plate and a new plate sealer applied. The plate was incubated for 60 minutes at room temperature on a horizontal orbital shaker set at 500±50 rpms. The Streptavidin Plate was washed four times with wash buffer and then excess wash buffer removed. Each well received 200 μL of anti-digoxigenin conjugate (21 mL of a polyclonal antibody against digoxigenin, conjugated to alkaline phosphatase) and the plate was covered with a new plate sealer. The plate was again incubated for 60 minutes on the shaker at a temperature of approximately 20-25° C. The Streptavidin Plate was washed six times with wash buffer and then excess wash buffer removed. 50 μL of substrate solution (lyophilized NADPH with stabilizers in a buffered solution) was added to each well and then the plate was covered with a new plate sealer. The plate was again incubated for 60 minutes on the shaker at a temperature of approximately 20-25° C. The plate was not washed. 50 μL of amplifier solution (buffered solution containing INT-violet with stabilizers) was added to each well and the plate covered with a new plate sealer. The samples were incubated for 30 minutes on the shaker at a temperature of approximately 20-25° C. and not washed. At the end of this time, 50 μL of a stop solution (2 N sulfuric acid) was added to each well. The optical density (OD) of each well was determined within 30 minutes, using a microplate reader set to 490 nm. If wavelength correction was available, the optical density was set to 650 nm or 690 nm. If wavelength correction was not available, readings at 650 nm or 690 nm were subtracted from the readings at 490 nm to correct for optical imperfections in the plate. Readings made directly at 490 nm without correction may be higher and, therefore, less accurate.
The results were calculated by determining the average of the duplicate optical density readings for each control and sample. The average negative control optical density value was subtracted from all average optical density values. The calculated positive control optical density value should be greater than or equal to 1.5. Results are recorded in Table 4.
EXAMPLE 4
Sample Preparation
A supemate sample of log phase growth BaF3 cells, a mouse hematopoietic cell line, was tested for possible mycoplasma contamination. For this experiment, 33 μL of supemate sample was diluted with 297 μL of Cell Lysis Diluent, vortexed and stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3. Results are recorded in Table 4.
EXAMPLE 5
Sample Preparation
A supemate sample of log phase growth HepG2 cells, a human hepatocellular carcinoma cell line, was tested for possible mycoplasma contamination. For this experiment, 33 μL of supemate sample was diluted with 297 μL of Cell Lysis Diluent, vortexed and stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3. Results are recorded in Table 4.
EXAMPLE 6
Sample Preparation
Log phase growth A431 cells, a human epidermoid carcinoma cell line, were tested for possible mycoplasma contamination. 5×105 cells were harvested and pelleted. The cell pellet was stored on ice until prepared for the assay. Alternatively, the pellet can be stored at ≦−20° C. for use at a later time. If stored at ≦−20° C., the pellet should be thawed on ice before prepared for the assay. Cell lysate samples were prepared using the following procedure: Four hundred microliters of Cell Lysis Diluent was added to the cell pellet containing 5×105 cells to obtain a final concentration of 1.25×106 cells per milliliter. The cells were pipetted up and down several times until resuspended and vortexed for 15 to 20 seconds. This cell lysate is further diluted 40- to 10-fold with Cell Lysis Diluent to obtain a final concentration of approximately 3×104 to approximately 1.2×105 cells per milliliter. In this experiment, 33 μL of cell lysate at 1.25×106 cells/mL was diluted with 297 μL of Cell Lysis Diluent to obtain a final concentration of 1.25×105 cells/mL. This sample was stored on ice until assayed. Alternatively, the cell lysate sample can be stored at ≦−20° C. for use in a later assay. If stored at ≦−20° C., the cell lysate sample should be thawed on ice before use in the assay.
Assay Procedure
The assay procedure was performed as described above in Example 3. Results are recorded in Table 4.
EXAMPLE 7
Sample Preparation
Log phase growth K562 cells, a human chronic myelogenous leukemia cell line, were tested for possible mycoplasma contamination. 5×105 cells were harvested and pelleted. The cell pellet was stored on ice until prepared for the assay. Cell lysate samples were prepared using the following procedure: Four hundred microliters of Cell Lysis Diluent was added to the cell pellet containing 5×105 cells to obtain a final concentration of 1.25×106 cells per milliliter. The cells were pipetted up and down several times until resuspended and vortexed for 15 to 20 seconds. This cell lysate was further diluted by adding 297 μL of Cell Lysis Diluent to 33 μL of cell lysate at 1.25×106 cells/mL to obtain a final concentration of 1.25×105 cells/mL. This sample was stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3. Results are recorded in Table 4.
EXAMPLE 8
Sample Preparation
A second culture of log phase growth K562 cells were tested for possible mycoplasma contamination. 5×105 cells were harvested and pelleted. The cell pellet was stored on ice until prepared for the assay. Cell lysate samples were prepared using the following procedure: Four hundred microliters of Cell Lysis Diluent was added to the cell pellet containing 5×105 cells to obtain a final concentration of 1.25×106 cells per milliliter. The cells were pipetted up and down several times until resuspended and vortexed for 15 to 20 seconds. This cell lysate was further diluted by adding 297 μL of Cell Lysis Diluent to 33 μL of cell lysate at 1.25×106 cells/mL to obtain a final concentration of 1.25×105 cells/mL. This sample was stored on ice until assayed.
Assay Procedure
The assay procedure was performed as described above in Example 3. Results are recorded in Table 4.











TABLE 4







Dilution or
OD Values



Sample
Sample Type
Cells/mL
(calculated)
Result







CTLL-2
Supernate
1:10
1.025
Positive


BaF3
Supernate
1:10
0.183
Positive


HepG2
Supernate
1:10
0.000
Negative


A431
Cell Lysate
1.2 × 105
1.042
Positive


K562
Cell Lysate
1.2 × 105
1.912
Positive


K562
Cell Lysate
1.2 × 105
0.005
Negative









EXAMPLE 9
The method and kit of the invention as described in Example 1 were compared to a standard Agar plating method and a PCR method using a mycoplasma detection kit available from ATCC (American Tissue Culture Collection, Monassas, VA). As can be seen from the results set forth in Table 5, the method of the invention, designated MycoProbe®, identified one sample missed by the PCR method.







TABLE 5







Mycoplasma Detection Methods Comparison










Mycoplasma
MycoProbe ®
PCR (ATCC)
Agar Plating





A. laidlawii
+
+
+


M. arginini
+
+
+


M. fermentans
+
+
+


M. hominis
+
+
+


M. hyorhinis
+
+
+


M. orale
+
+
+


M. pirum
+
−
+


M. salivarum
+
+
+





A = Acholeplasma






EXAMPLE 10
Assay cross-reactivity of the method and kit described in Example 1 was tested using 1×104 CFU/well of microbes and 15,000 cells/well of mammalian cells listed below in Table 6: Cross-reactivity. Results were determined using the OD levels indicated in Table 2. This assay recognized the eight mycoplasma species previously listed in the Sensitivity Table (Table 2) and two closely related prokaryote species (based on 16S rRNA homology), Ureaplasma (U.) urealyticum and Lactobacillus (L.) casei. U. urealyticum is a mycoplasma associated with human urogenital diseases and is not found usually as a cell culture contaminant. U. urealyticum was detectable using as few as 2.7×103 CFU/well. L. casei is a lactic acid fermenting bacteria that is not found as a cell culture contaminant. No significant cross-reactivity was observed for other microbes in the panel. Mammalian cells did not show cross-reactivity or interference when tested using the recommended concentration range.







TABLE 6







Cross-Reactivity










Organism
Classification
Result
OD





Ureaplasma urealyticum
Mollicute (mycoplasma)
Positive
0.168


Lactobacillus casei
Gram Positive Bacteria
Positive
1.748


Bacillus subtilis
Gram Positive Bacteria
Negative
0.045


Escherichia coli
Gram Negative Bacteria
Negative
0.009


Torulopsis candida
Yeast
Negative
0.004


Cryptococcus albidus
Yeast
Negative
0.001


Geotrichum sp.
Mold
Negative
0.002


Penicillium sp.
Mold
Negative
0.002


Cladosporium sp.
Mold
Negative
0.002


Human
K562
Negative
0.014


Mouse
EL-4
Negative
0.000


Rat
NR-8383
Negative
0.000

## Claims

1. A method for detecting the presence of mycoplasma in a sample comprising: a) contacting a sample treated to release ribosomal RNA of mycoplasma present in the sample with two or more first oligonucleotide probes wherein each first oligonucleotide probe is substantially complementary to a portion of a 16S ribosomal RNA of at least one mycoplasma species and is labeled with a capture ligand, the first oligonucleotide probes comprising SEQ ID NO's 14 and 15; b) contacting the sample with two or more second oligonucleotide probes wherein each second oligonucleotide probe is substantially complementary to a different portion of the 16S ribosomal RNA of the at least one mycoplasma species and is labeled with a first component of a detection system, the first component comprising a detection ligand, the second oligonucleotide probes comprising SEQ ID NO's 5 and 10; c) incubating the sample with the first and second oligonucleotide probes under hybridization conditions to form a hybridization solution and for a time sufficient for the probes to hybridize to 16S ribosomal RNA of mycoplasma species present in the sample; d) contacting the hybridization solution with a solid phase coated with a capture receptor capable of specifically binding to the capture ligand of the first labeled oligonucleotide probe(s); and e) detecting the presence of mycoplasma ribosomal RNA in the sample with a detection solution comprising a labeled detection receptor.

2. The method of claim 1 wherein the capture ligand is biotin.

3. The method of claim 2 wherein the capture receptor on the solid phase is streptavidin or anti-biotin.

4. The method of claim 1 wherein the presence of mycoplasma ribosomal RNA in the sample is detected by detecting the presence of the detection ligand on the second oligonucleotide probe(s) bound to mycoplasma ribosomal RNA present in the sample.

5. The method of claim 4 wherein the detection receptor is labeled with a signal generating moiety and wherein the detection receptor is chosen to specifically bind to the detection ligand, further comprising reacting the signal generating moiety with a substrate solution to produce a detectable signal.

6. The method of claim 5 wherein the detection ligand is digoxigenin and the detection receptor is anti-digoxigenin antibody.

7. The method of claim 1 wherein the presence of two or more species of mycoplasma in the sample is detected.

8. The method of claim 7 wherein the species of mycoplasma detected are selected from the group consisting of Mycoplasma arginini, Mycoplasma orale; Mycoplasma fermentans; Mycoplasma hyorhinis; Mycoplasma pirum; Mycoplasma hominis; and Mycoplasma salivarium.

9. The method of claim 5 wherein the signal generating moiety is alkaline phosphatase.

10. The method of claim 9 wherein the detectable signal produced is colorimetric.

11. The method of claim 9 wherein an amplifying solution is added that amplifies the detectable signal.

12. The method of claim 1 wherein the first oligonucleotide probes further comprise SEQ ID NOs. 12 and 13 and wherein the second oligonucleotide probes further comprise SEQ ID NOs. 1 to 4, 6 to 9 and 11.

13. A mycoplasma detection kit for detecting the presence of mycoplasma contamination comprising:
a) two or more different first oligonucleotide probes comprising SEQ. ID NOs. 14 and 15 wherein each first oligonucleotide probe is substantially complementary to a portion of a 16S ribosomal RNA of one or more mycoplasma species and is labeled with a capture ligand;
b) two or more different second oligonucleotide probes comprising SEQ. ID NOs. 5 and 10 wherein each second oligonucleotide probe is substantially complementary to a different portion of the 16S ribosomal RNA of the mycoplasma species than any of the first oligonucleotide probes and each is labeled with a first component of a detection system, wherein the first component comprises a detection ligand;
c) a solid phase coated with a capture receptor chosen to specifically bind to the capture ligand on the first probes; and
d) a detection solution comprising a second component of the detection system, wherein the second component comprises a labeled detection receptor.

14. The mycoplasma detection kit of claim 13 further comprising each of the first oligonucleotide probes of SEQ ID NOs. 12 and 13 and each of the second oligonucleotide probes of SEQ ID NOs. 1 to 4, 6 to 9 and 11.

15. The mycoplasma detection kit of claim 13 wherein the capture ligand is biotin.

16. The mycoplasma detection kit of claim 13 wherein the detection ligand is digoxigenin and the detection receptor is anti-digoxigenin antibody.

17. The mycoplasma detection kit of claim 13 wherein the kit is capable of detecting the presence of two or more species of mycoplasma in a sample.

18. The mycoplasma detection kit of claim 17 wherein the species of mycoplasma detected are selected from the group consisting of Mycoplasma arginini, Mycoplasma orale; Mycoplasma fermentans; Mycoplasma hyorhinis; Mycoplasma pirum; Mycoplasma hominis; and Mycoplasma salivarium.

19. The mycoplasma detection kit of claim 13 wherein the detection receptor is labeled with a signal generating moiety and wherein the detection receptor specifically binds to the detection ligand.

20. The mycoplasma detection kit of claim 19 wherein the signal generating moiety is alkaline phosphatase.

21. The mycoplasma detection kit of claim 19 further comprising an amplifying solution to amplify a detectable signal.

22. The mycoplasma detection kit of claim 13 further comprising one or more first oligonucleotide probes selected from the group consisting of SEQ ID NOs. 12 and 13.

23. The mycoplasma detection kit of claim 13 further comprising one or more second oligonucleotide probes selected from the group consisting of SEQ ID NOs. 1 to 4, 6 to 9 and 11.