The Universal Mycoplasma Detection Kit offers a quick and sensitive PCR-based test to detect mycoplasma contaminants in cell culture. All components required for the PCR reaction are provided and have been optimized for amplification. High specificity is obtained through the utilization of a proprietary mix of buffers, dNTPs and thermostable polymerase, combined with universal primers that are specific to the 16S rRNA coding region in the mycoplasma genome. DNA originating from other sources, such as E.coli and human, mouse, hamster, herring sperm, dog or rat genomic DNA, are not amplified. A touchdown PCR regimen increases sensitivity of the assay, along with enhancing specificity.

• Touchdown PCR was applied by Eldering et al. in 2004 (6)
• PCR with the Universal primers and touchdown is the method used in publications by Roche, Bayer, Genentech, Medimmune and others (1)
• The kit is designed to meet European Pharmacopeia 2.6.7 guidelines (4)

 

The Universal Mycoplasma Detection Kit recognizes over 60 species of Mycoplasma, Acholeplasma, Spiroplasma and Ureaplasma including the eight species most likely to afflict cell cultures: M. arginini, M. fermentans, M. hominis, M.hyorhinis, M. orale, M. pirum, M. salivarium, and A. laidlawii. Samples that are positive for mycoplasma are easily recognized by a distinct PCR product ranging in size (depending on the species) from 434 to 468 bp on an agarose gel.

See Sensitivity and Specificity Data for a complete listing of the species detected with the Universal Mycoplasma Detection Kit.

Figure 1 – Basic Procedure
Figure 2 – Sample Preparation Makes a Difference
Figure 3 – Choice of Amplification Enzyme Makes a Difference

Table 1 – Sensitivity (LOD) of All Mycoplasma Species Detected
Figure 4 – M.arginini titration
Figure 5 – Touchdown vs Standard PCR

Supression of false positive signals
Extensive testing of samples without target demonstrated occasional amplification of environmental DNA from bacteria whose sequences most closely match “uncultured bacteria”. These products were usually very faint but sometimes were as strong as the products in Figure 5 lanes 6 above when amplification was for 40 cycles. These signals are less prevalent when the PCR was assembled in a laminar flow hood, and these products were not seen in both samples when duplicate samples were analyzed. To suppress these signals further we suggest an amplification protocol of 32 cycles instead of 40. At 32 cycles, there is some diminution in the sensitivity of the assay, but less than 20 genomes of most Mycoplasma species is still detected. Amplification for 40 cycles will achieve the highest level of sensitivity but occasional amplification of environmental DNA should be expected unless extreme precautions are taken such as dedicated laboratories with controlled airflow, controlled workflow, positive displacement pipettes and frequent decontamination procedures.

Background on Mycoplasmas

Mycoplasmas are small, self-replicating bacteria that are widely recognized as serious contaminants in cell cultures. These microorganisms range from 0.2 to 0.8 µm and are classified as Mollicutes due to the absence of a rigid cell wall. They are able to grow to cell densities in the range of 10⁷-10⁸ organisms per mL without any obvious change in the cell culture medium such as turbidity or pH shifts. Mycoplasmas have a very small genome, ranging from 580 kb to 2,220 kb, resulting in limited metabolic pathways and parasitic characteristics (15).

However, mycoplasmas have a high percentage of conserved genes and a relatively low GC content that are useful in their detection. The Universal Primers are very specific for the detection of mycoplasma due to exact or nearly exact matches within the 16S rRNA region of the mycoplasma genome. Other genomes, such as E.coli, are not recognized due to significant mismatches between base pairs of the primer sequences and genomic DNA.

Prevalence of Mycoplasma Contamination

Mycoplasmas are frequent contaminants of cell cultures. Numerous studies have been published that establish mycoplasma contamination of continuous cell cultures in the range of 15-35%, with primary cell cultures exhibiting a 1% contamination rate (5, 13). Since mycoplasma contamination is often unrecognized, these rates are often estimated to be even higher.

There are over 190 species of mycoplasma, but only 20 distinct species, of human, bovine and porcine origin, have been identified in cell culture (2, 16). Of those twenty, eight species account for approximately 95% of all mycoplasma contamination in cell culture:

• Mycoplasma arginini (bovine)
• Mycoplasma fermentans (human)
• Mycoplasma hominis (human)
• Mycoplasma hyorhinis (porcine)
• Mycoplasma orale (human)
• Mycoplasma pirum (human)
• Mycoplasma salivarium (human)
• Acholeplasma laidlawii (bovine)

 

Sources of Mycoplasma Contamination

Despite having limited metabolic pathways and no cell wall, mycoplasmas are resilient. Mycoplasmas are dispersed by aerosol droplets or particles generated while pipetting and handling media and different cell types simultaneously. Possible sources of mycoplasma contamination are (5):

• Infected incoming cells (cross-contamination)
• Cell culture media, sera or trypsin
• Laboratory personnel

The primary source of mycoplasma contamination is cross contamination from infected cell cultures. Laboratory personnel are also a key source of contamination, as human mycoplasmas (M.orale, M. fermentans and M. salivarium) are major species detected in cell cultures. Laboratory equipment, benches and flow hoods have also been identified as sources of contamination, along with cell culture reagents such as media and serum.

To reduce the risk of mycoplasma contamination, test all incoming cells and routinely screen cell cultures and other sources of infection. Aseptic cell culture techniques are critical and only one cell culture should be processed at a time. Antibiotics should not be used in lieu of stringent cell culture procedures (see Antibiotics and Mycoplasma section).

Effect of Mycoplasma in Cell Cultures

Mycoplasma contamination can cause a wide variety of adverse effects on the function and activities of cells in culture. Mycoplasmas such as M. orale and M. arginini, for example, deaminate the amino acid arginine as an alternate energy source, depleting culture arginine, resulting in limited cell growth and possibly leading to chromosome damage (7). A. laidlawii and M. hyorhinis ferment sugars in media, resulting in a decrease in pH. Most mycoplasmas create these subtle, but often significant effects, leading to misinterpretation of results and compromising the validity of data generated for research and/or development projects. Consequences of mycoplasma contamination include (5,12,16):

• inhibition of cell metabolism
• induction of chromosomal abnormalities
• disruption of DNA and RNA synthesis
• changes in virus and antibody production
• interference with growth rate of cells
• Depletion of arginine
• Change in pH
• Activation of B cells
• Alter gene expression in cells

Figure 6. charts the 'Unpredictable Effect of Mycoplasma Infection on Transfection'.

Antibiotics and Mycoplasma

Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack a cell wall. Streptomycin inhibits about half the mycoplasma strains and is ineffective against many strains. Gentamycin is generally ineffective at the concentrations routinely used in cell culture. In fact, mycoplasmas are generally resistant to antibiotic mixtures that are commonly used in cell cultures (5).

Mycoplasma detection can be slightly improved by growing cells for two or more passages in antibiotic-free medium; however this is highly dependent on the antibiotic and species. Antibiotic-free medium is not required for testing with the Universal Mycoplasma Detection Kit.

In general, the use of antibiotics is not recommended in cell cultures except for specific applications and for a limited time. Routine antibiotic use may result in proliferation of antibiotic-resistant organisms, and may allow low level infections to remain undetected for long periods of time (10, 11).

Antibiotics have been considered for the removal of mycoplasma from contaminated cultures. Primary tissues and new cell lines derived from patients can be infected with mycoplasma and there may be a compelling need to rid these cultures of the mycoplasma contamination. These procedures can be successful but should not be undertaken lightly. For more information, please refer to Borup-Christensen’s article on “Curing human hybridomas infected with Mycoplasma hyorhinis” (3) included in References and Further Reading.

1. Asarnow, D. et al. Validation and international regulatory experience for a mycoplasma touchdown PCR assay. Biologicals 38: 224-231 (2010).
2. Barile, M. F., Hopps, H. E., Grabowski, M. W., Riggs, D. B. and Del Giudice, R. A. The Identification and Sources of Mycoplasmas Isolated from Contaminated Cell Cultures. Ann. NY Acad. Sci. 225:251-264 (1973).
3. Borup-Christensen, P., Erb, K. and Jensenius, J. C. Curing human hybridomas infected with Mycoplasma hyorhinis. Journal of Immunological Methods 110 (2): 237-240 (1988).
4. Deutschmann, S. M., Kavermann, H. and Knack, Y. Validation of a NAT-based Mycoplasma assay according to European Pharmacopoiea. Biologicals 38: 238-248 (2010).
5. Drexler, H.G. and Uphoff, C.C. Mycoplasma contamination of cell cultures: incidence, sources, effect, detection, elimination, prevention. Cytotechnology 39:75-90 (2002).
6. Eldering, J. A., Felten, C., Veilleux, C. A. and Potts, B. J. Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins. Biologicals 32: 183-193 (2004).
7. Freed, J. J. and Schatz, S. A. Chromosome aberrations in cultured cells deprived of single essential amino acids. Exp. Cell Res. 55:393-409 (1969).
8. Johansson, K. E. and Pettersson, B. Taxonomy of Mollicutes In Molecular Biology and Pathogenicity of Mycoplasmas, S. Razin and R. Herrmann, eds. (Kluwer Academic/Plenum., New York, 2002) p. 1–30.
9. Krass, C. J. and Gardner M.W. Etymology of the Term Mycoplasma. Int. J. of Syst. Bact. 23 (1): 62-64 (1973).
10. Lincoln, K. L., and Gabridge, M. G. Cell Culture Contamination: Sources, Consequences, Prevention and Elimination. In Methods in Cell Biology, Vol. 57 (Academic Press, New York, 1998) p. 49-65.
11. McGarrity, G. J. Spread and Control of Mycoplasmal Infection of Cell Cultures. In Vitro 12:643-647 (1976).
12. McGarrity G. J., Vanaman, V. and Sarama, J. Cytogenetic Effects of Mycoplasmal Infection of Cell Cultures: A Review. In Vitro 20 (1):1-18. (1984).
13. McGarrity G. J., Steiner, T. and Vanaman, V. Detection of Mycoplasmal Infection of Cell Cultures by DNA Fluorochrome Staining. In Methods in Mycoplasmology, Vol. 2, J. G. Tully and S. Razin, eds. (Academic Press, New York, 1983) pp. 183-190.
14. Perlman, D., Rahman, S. and Semar, J. Antibiotic Control of Mycoplasma in Tissue Culture. Applied Microbiology 15 (1):82-85 (1967).
15. Razin S., Yogev D., and Naot Y. Molecular Biology and Pathogenicity of Mycoplasmas. Microbiol Rev 63: 1094–1156 (1998).
16. Rottem, Shlomo. Interaction of Mycoplasmas With Host Cells. Physiol Rev 83: 417–432 (2003).

1.  Why should I test my cell cultures for mycoplasma?

    

2.  What is the sensitivity of the Universal Mycoplasma Detection Kit?

    

3.  How can I increase the sensitivity of the Universal Mycoplasma Detection Detection Kit?

    

4.  Does the kit detect DNA from species other than mycoplasma?

    

5.   How long does it take to get results?

    

6.  Can the assay be used to identify which mycoplasma species contaminated my cells?

    

7.   Which species of mycoplasma does this kit recognize?

    

8.  Can mycoplasma be eliminated from my cells?

    

9.  Can I test a cell culture supernatant for mycoplasma instead of a cell lysate?

    

10.  Why is it recommended to centrifuge samples at 4°C instead of room temperature?

     

11. ; Can media be tested with the Universal Mycoplasma Detection Kit?

     

12.  Can either adherent cells or suspension cells be used in the assay?

     

13.  Will the presence of antibiotics in the culture medium affect the assay?

     

14.  Should I harvest the adherent cells with trypsin?

     

15.  My cell pellet is difficult to resuspend. What should I do?

     

16.  I used a water bath for the inactivation step at 95°C, and the water began to boil. Will this affect my samples?

     

17.  The lids of the lysis tubes were a little loose after the heat inactivation step. Is this okay?

     

18.  If I have to interrupt the procedure, how should I store my samples?

     

19.  Is PCR an accepted method for mycoplasma detection?

     

20.  Are there any special precautions to avoid cross contamination when performing PCR?

     

21.  What is the likelihood of observing false positive results?

     

22.  Is Taq DNA Polymerase included in the kit?

     

23.  What primers are used in the assay?

     

24.  Is it necessary to use the reaction tubes included in the kit?

     

25.  What is touchdown PCR? Why change the annealing temperature during cycling?

     

26.  What will happen if I don’t use Touchdown PCR?

     

27.  Do I have to follow the exact touchdown procedure described in the protocol?

     

28.  What controls are included in the kit?

     

29. &Can I prepare 50-µL reactions instead of 25 µL for PCR?

     

30.  I prepared duplicate samples for PCR. What should I do if one sample is positive, but the other sample is negative for mycoplasma contamination?

     

31.  My negative control shows a faint band in the 434 bp to 468 bp range. What should I do?

     

32.  How is this kit different from ATCC’s former Mycoplasma Detection Kit?

     

33.  I inadvertently skipped the inactivation step in the sample preparation. Will the assay still work?

     

34.  Can I store the kit at 4°C instead of going through numerous freezing and thawing at 20°C?
    
    
    1. Why should I test my cell cultures for mycoplasma?
       
       Since mycoplasma is invisible under normal microscopic viewing, routine screening of cell lines for mycoplasma contamination is essential for reliable results. Mycoplasma contamination is a common and widespread problem that can cause subtle and adverse effects on cells in culture. Mycoplasma can inhibit cell metabolism, alter cell growth and morphology, disrupt DNA and RNA synthesis, cause chromosomal abnormalities and affect virus yields. Mycoplasma contamination compromises the safety and efficacy of any research and/or development project.

        

    2.  What is the sensitivity of the Universal Mycoplasma Detection Kit?
       
       The kit detects less than 20 genomes of M.arginini in a standard assay with 32 cycles of amplification.. The range of detection varies depending on species, but, in general, the kit detects 5 to 25 femtograms DNA, or 4 to 40 genomes, of the eight species that are responsible for 95% of all mycoplasma contamination in cell culture. Infected cultures typically generate robust signals after 32 cycles. To avoid false positive signals from environmental bacteria, we recommend that 32 cycles of PCR be used for routine screening.

        

    3.  How can I increase the sensitivity of the Universal Mycoplasma Detection Kit?
       
       The number of amplification cycles can be increased to 40 cycles. This is done by increasing the number of cycles in the second half of the Touchdown protocol from 12 to 20 cycles. Detection results for over 60 strains of mycoplasma using a 40 cycle protocol can be found under Sensitivity and Specificity Data on the Universal Mycoplasma Detection Kit product page. Laboratories with positive displacement pipettes and strict control of environmental conditions may use 40 cycles to attain these levels of sensitivity. Even with the best control, occasional amplifications of environmental DNA may be detected after 40 cycles. The identity of these amplicons can be established by sequencing.

        

    4.  Does the kit detect DNA from species other than mycoplasma?
       
       The test is negative for E. coli, Chlamydia, human, mouse, hamster, herring sperm, dog, and rat genomic DNAs. The primers are able to amplify the rRNA genes of gram positive bacteria such as Bacillus and Staphylococcus. The primers are more efficient at amplifying rRNA genes from some environmental (“uncultured”) bacteria that may or may not be present in your laboratory. For this reason, care must be taken to avoid environmental contamination of the sample. At ATCC, we assemble the PCR reactions in a laminar flow hood.

        

    5.  How long does it take to get results?
       
       Total time from sample preparation to results is less than 3 hours.

        

    6. Can the assay be used to identify which mycoplasma species contaminated my cells?
       
       A positive mycoplasma results in a specific band following agarose gel electrophoresis. If speciation is required, the band can be excised from the gel and sequenced. This will identify the contaminant as an avian, porcine, equine, or human type, but the exact species may not be unambiguously determined from a 16S rRNA sequence.

        

    7. Which species of mycoplasma does this kit recognize?
       
       The assay detects over 60 unique strains of mycoplasma including the eight species most likely to afflict cell cultures: M. arginini, M. fermentans, M. hominis, M.hyorhinis, M. orale, M. pirum, M. salivarium, and A. laidlawii. A complete list of the species detected by the assay can be found under Sensitivity and Specificity Data on the Universal Mycoplasma Detection Kit product page.

        

    8. Can mycoplasma be eliminated from my cells?
       
       The most reliable procedure is to discard the contaminated cultures and obtain a fresh culture. If this is not feasible, then the cultures can be treated with antibiotic regimens. Since mycoplasma are resistant to antibiotics such as penicillin, Tiamutin, Minocycline, and Ciprofloxacin-type antibiotics are often used in short, alternating treatments. For surface contamination, Benzylammonium compounds are a safe and effective treatment.

        

    9. Can I test a cell culture supernatant for mycoplasma instead of a cell lysate?
       
       Since mycoplasma are generally cell-associated, the most sensitive test involves a cell pellet. For best results, start with a cell pellet and prepare the sample according to the recommended protocol. In some cases, however, it is not practical to sacrifice a flask in order to perform the test. In this event, a 1 ml culture supernatant may be centrifuged as described in the protocol, and the pellet treated with the lysis mix. The test will not be optimal, but is still a very sensitive test for the presence of mycoplasma contaminants.

        

    10. Why is it recommended to centrifuge samples at 4°C instead of room temperature?
        
        Most centrifuges generate heat, which causes cells to lyse. If cells lysis occurs during centrifugation, mycoplasma-derived nucleases may degrade the sample DNA. Spinning at 4C reduces the potential for cell lysis during centrifugation.

         

    11. Can media be tested with the Universal Mycoplasma Detection Kit?
        
        The Universal Mycoplasma Detection Kit was not designed to test media. It may be used to test media, but sample size may be critical. Media that are lightly contaminated may not contain enough organisms in 1 mL to be detected in the PCR reaction. It may be necessary to centrifuge more than 1 mL of medium at 13,000 rpm prior to resuspending in a 50-µL aliquot.

         

    12. Can either adherent cells or suspension cells be used in the assay?
        
        Either adherent or suspension cells can be tested. Adherent cells should be harvested at 50-70% confluency and suspension cells should be harvested in log-phase growth.

         

    13. Will the presence of antibiotics in the culture medium affect the assay?
        
        Although most common antibiotics will not eliminate mycoplasma, the presence of antibiotics may reduce the sensitivity of the kit by artificially suppressing levels of detectable mycoplasma. Mycoplasma detection may be improved by growing cells for two or more passages in antibiotic-free medium. However this depends on the antibiotic and species. Penicillin, for example, has no effect since mycoplasma lack a cell wall. In fact, mycoplasmas are generally resistant to the penicillin-streptomycin mixtures that are commonly used in cell cultures. For more information, see the Antibiotics and Mycoplasma section on the Universal Mycoplasma Detection Kit product page.

         

    14. Should I harvest the adherent cells with trypsin?
        
        Adherent cells should not be treated with trypsin or EDTA as these agents disrupt mycoplasma. Harvest by scraping cells into medium or PBS.

         

    15. My cell pellet is difficult to resuspend. What should I do?
        
        The pellet may not dissolve easily in the Lysis Buffer. The incubation step at 37°C should help to dissolve the pellet. After the incubation, vortex the pellets briefly to resuspend. Vortexing the samples will not shear the DNA. If the pellet is viscous or still difficult to resuspend, more Lysis Buffer can be added. The results will not be compromised, since the difficulty is most likely due to a large number of cells in the sample.

         

    16. I used a water bath for the inactivation step at 95°C, and the water began to boil. Will this affect my samples?
        
        The samples will be fine even if the incubation temperature reaches 100°C for a substantial amount of time.

         

    17. The lids of the lysis tubes were a little loose after the heat inactivation step. Is this okay?
        
        When using a water bath for the inactivation step, the tops of the tubes may become hot and loosen. After removing the tubes from the water bath, allow the tubes to cool before further handling.

         

    18. If I have to interrupt the procedure, how should I store my samples?
        
        Samples can be stored (1) after collecting the cell pellet and discarding the media, (2) after the digestion step, and (3) after the heat inactivation step. The cell pellets or lysates can be stored at -80°C. The samples can also be stored at -80°C after the PCR step.

         

    19. Is PCR an accepted method for mycoplasma detection?
        
        PCR is gaining acceptance world-wide as a viable option for mycoplasma detection. Our kit is designed to meet European Pharmacopeia 2.6.7 guidelines (Biologicals 38, March 2010).

         

    20. Are there any special precautions to avoid cross contamination when performing PCR?
        
        Sample preparation, amplification and detection should occur in separate areas and use dedicated equipment. Keep reactions and components capped as much as possible. Use pipette tips with hydrophobic filters to avoid cross-contamination with DNA. Work in a PCR hood with laminar flow or a laminar flow hood. Pipette tips, gels, and electrophoresis buffer that come into contact with post-PCR products should be disposed of carefully to avoid contamination with previously generated PCR fragments.

         

    21. What is the likelihood of observing false positive results?
        
        Extensive testing of samples without target demonstrated occasional amplification of environmental DNA from bacteria whose sequences most closely match “uncultured bacteria”. These products were suppressed when amplification was assembled in a laminar flow hood, and these products were not seen in both samples when duplicate samples were analyzed. The 32-cycle PCR protocol is recommended to avoid the generation of false positive results.
        
        Amplification of previously generated PCR products is a second way to generate a false positive. For this reason, care should be taken in the handling of post-PCR products, including tips, electrophoresis buffer, and agarose gels that contain PCR products. At ATCC, 8% bleach in water is used to decontaminate surfaces, and tips containing post-PCR products are ejected into a beaker filled with 8% bleach.

         

    22. Is Taq DNA Polymerase included in the kit?
        
        The kit contains all the components required for sample preparation and detection by PCR. A thermostable polymerase is a component of the Universal PCR Mix. This proprietary mixture also includes dNTPs, buffers, and MgCl2, formulated for optimal amplification. Universal primers are also included in the kit.

         

    23. What primers are used in the assay?
        
        Universal forward and reverse primers are a component of the kit. The primers target the highly conserved 16S rRNA region of the mycoplasma genome and generate a specific PCR product ranging from 434 bp to 468 bp.

         

    24. Is it necessary to use the reaction tubes included in the kit?
        
        It is recommended that the tubes included in the kit are used when setting up the reactions. ATCC tested tubes from various sources and determined that many tubes tend to pop open during the sample preparation heat inactivation step. The tubes are provided to prevent any cross contamination.

         

    25. What is touchdown PCR? Why change the annealing temperature during cycling?
        
        Touchdown PCR (TD-PCR) is a PCR cycling program that has been demonstrated to increase specificity and sensitivity by varying the annealing temperature during amplification. In the initial cycles, the annealing temperature is higher than the target optimum (projected melting temperature of the primers used). The temperature is then decreased by 0.5 to 2°C increments in subsequent cycles until the optimal target temperature is reached. The target or “touchdown” annealing temperature is then used for the remaining cycles. TD-PCR helps ensure the accuracy of your results.

         

    26. What will happen if I don’t use touchdown PCR?
        
        If you use regular PCR instead of the touchdown procedure, non-specific amplification may occur and bands may be present that are not derived from mycoplasma DNA, but from the cellular DNA in the extract.

         

    27. Do I have to follow the exact touchdown procedure described in the protocol?
        
        No. Decreasing the temperature by 1°C every 2 cycles is acceptable.

         

    28. What controls are included in the kit?
        
        A positive control (pUC19::M. arginini target) is included to verify that the PCR reaction was successful. A negative control is prepared by combining the Universal PCR Mix with H₂O or TE buffer. We also recommend that a second positive control is prepared which combines the positive control with a cell lysate to confirm that the cell lysate did not inhibit the PCR reaction. Use of more than 10⁶ cells per reaction may inhibit PCR.

         

    29. Can I prepare 50-µL reactions instead of 25 µL for PCR?
        
        Yes, just double the amount added of each component in the PCR reaction.

         

    30. I prepared duplicate samples for PCR. What should I do if one sample is positive, but the other sample is negative for mycoplasma contamination?
        
        Duplicate samples that show conflicting results (e.g., a 464-bp band is observed in one lane but not the other) may indicate a low level of contamination in your test sample. Prepare six duplicate samples and repeat the PCR to verify the result. Growth of the cells in the absence of antibiotic may increase the signal. Sequencing the PCR product is recommended in borderline cases, since some non-mycoplasma bacterial DNA targets can generate a positive signal.

         

    31. My negative control shows a faint band in the 434 bp to 468 bp range. What should I do?
        
        The presence of a 434-bp to 464-bp band in the negative control lane is most likely due to cross contamination during PCR sample preparation. Prepare new samples and repeat PCR. If the problem persists, please contact ATCC Technical Service.

         

    32. How is this kit different from ATCC’s former Mycoplasma Detection Kit?
        
        The Universal Mycoplasma Detection Kit components are formulated as a mix, and include all the components required for PCR. Unlike the previous Mycoplasma Detection Kit, a thermostable DNA Polymerase is included in the kit, and the reaction buffers and conditions have been optimized for amplification using this enzyme. The new kit has increased sensitivity, convenience, a straightforward protocol and a shorter time to results. The former kit was capable of speciation since the PCR product was from a nonconserved spacer region in the 16-23S rRNA operon.

         

    33. I inadvertently skipped the inactivation step in the sample preparation. Will the assay still work?
        
        The assay will NOT work. The protease in the Lysis Mix must be inactivated prior to adding the lysate to the PCR mixture.

         

    34. Can I store the kit at 4°C instead of going through numerous freezing and thawing at 20°C?
        
        The kit may be stored at 4°C for short periods (a day or two) but prolonged storage at 4°C is not recommended because the protease in the Lysis Mix will start to degrade and the Universal PCR Mix will slowly lose potency.

You can order the Universal Mycoplasma Detection Kit from our online catalog if you are logged in and have an ATCC account associated with your login. Or call 800-638-6597 in the United States, and Puerto Rico or 703-365-2700 elsewhere. Customers in Europe, Australia, Canada, China, Hong Kong, India, Israel, Japan, Korea, Macau, Mexico, New Zealand, Singapore, and Taiwan, R.O.C. must order from our official distributors.