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Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
Mycoplasma Detection Kit-QuickTest
• Perform all assay steps at room temperature (22-28°C).
• Do not use H2O as the negative control.
• Be sure to fully mix samples after addition of reaction buffer.
Components
Description
StorageStored at 4 ~ 25°C for up to 12 months.
1. Negative control: the same unused cell culture medium (without mycoplasma contamination).2. Test samples: cell culture supernatant.
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Open the test plates.
Add 40 uL Reaction Buffer A to the sample wells.
In the first well, add 10 uL of the negative control. In subsequent wells, add 10 uL of the test samples or positive controls.Note: Negative control should be the cell culture media incubated the same as cells.
Shake gently, then let stand at room temperature for 5 min.
When a cell culture system is contaminated by mycoplasma, its metabolic enzymes will degrade culture medium components and produce metabolic products which are secreted into the cell culture supernatant. The presence of these metabolites indicates the presence of mycoplasma, as eukaryotic cells or bacteria will not produce these metabolites.
If the test sample contains the metabolites, the reaction system (including the test sample, the reaction sample well, and the reaction buffer) will turn green in color. The concentration of metabolites produced is proportional to how dark the color is, directly indicating the amount of mycoplasma in each sample. If there is no metabolite present, the reaction result (color) will be the same as the negative control, showing no mycoplasma.
Perform all steps at room temperature: 22 - 28°C.
Protocol① Add 40 ul Reaction Buffer A ④ Add 40 ul
Reaction Buffer B② Add 10 ul sample
③ oscillate gently
stand at room temperature for
5 min
⑤ oscillate gentlystand at room
temperature for 4 min
5 min 9 min
⑦ analyzethe colorchange
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Important Notes1.
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As the method is based on detecting metabolic products of mycoplasma, we recommend performing the test after 36-48 h continuous cell culture.
The optimal reaction conditions are at room temperature: 22-28°C. The reaction temperature must be over 18°C. We recommend warming the kit to room temperature before starting the experiment, especially in cold environment.
For most samples, we recommend batch testing. Please set up independent negative control samples for each batch to ensure that color reaction timing is controlled for different wells.
Notice
Contents Cat#: B39032 Cat#: B39035 Cat#: B39038
Test Wells
Reaction Buffer A
Reaction Buffer B
Stop Solution
Positive Control *
80 (4 test x 20)
5 mL
5 mL
0.4 mL
0.2 mL
400 (4 test x 100)
5 mL x 5
5 mL x 5
0.4 mL x 5
0.2 mL x 5
4000 (4 test x 1000)
5 mL x 50
5 mL x 50
0.4 mL x 50
0.2 mL x 50
* Positive Control is the metabolites of mycoplasma, not mycoplasma organism.
Add 40 uL Reaction Buffer B to all sample wells.
Shake gently, then let stand at room temperature for 4 min.
Add 5 uL Stop Solution, shake gently.
Monitor sample color changes within 30 min.
a) If the color of the test well is darker than the negative control well, the sample is positive for mycoplasma.
b) If the color of the test well is the same as the negative control well, the sample is negative for mycoplasma.
⑥ Add 5 ul Stop Solution
5. The following samples can NOT be examined by this method: a) Any kind of serum-free media ( such as Corning KBM851) ; b) F12 cell culture medium; c) DMEM/F12 cell culture medium.
These media contain high concentration of interfering components. These components can inherently cause color changing, leading to false results.
6. For research use only. Cannot be used for clinical purpose.
The material used to coat the test plate may change color when exposed to air. Only open the test plate immediately prior to use.
Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
How can I test if the fresh cell culture
medium is contaminated by
mycoplasma?
For the same sample, why are the results of
PCR methods positive, while the
results of this detection kit are
negative?
For the same sample, why are the results of
PCR methods negative, while the
results of this detection kit are
positive?
Why is it not recommended to use
H2O as negative control?
Can this mycoplasma detection kit be used
to detect contamination of cell culture supernatants stored at 4°C which
were collected in batch?
Fig A. Test results from Lab 1, the sample A and B were not contaminated by mycoplasma. Fig B. Test results from Lab 2, the sample C and D were contaminated by mycoplasma. Fig C. Test results from Lab 3, the sample E and F were contaminated by mycoplasma. The mycoplasma contamination of Sample E was more severe than Sample F.
1. This kit cannot distinguish between species of mycoplasma, but can effectively detect all types of mycoplasma.
2. If the cell culture system is contaminated by trace mycoplasma (less than 10 mycoplasma copies / uL cell culture supernatant), the result may show weakly positive. We suggest re-testing the mycoplasma contamination after appropriate extension of cell culture time (24-48 h).
Culture the fresh cell culture medium by cell culture flask in a CO2 incubator for 48 hours (sample A). Take fresh cell culture medium stored in a refrigerator as the control, and then test these samples with our kit. Compared to the control test well, if the color of sample A is deeper, the cell culture medium is contaminated by mycoplasma. If the colors are similar, the cell culture medium is not contaminated by mycoplasma. We recommend culturing the fresh cell culture medium by cell culture flask in a CO2
incubator 48 hours before testing the cell culture supernatant, in order to exclude the existence of mycoplasma contamination of the fresh cell culture medium.
Even if the mycoplasma is dead, the results of PCR method will come out as positive, while the results of this detection kit are negative, for the following reasons: PCR methods detect the existence of mycoplasma via amplify 16S rRNA sequences. Whether the mycoplasma is dead or alive, once there is the presence of mycoplasma 16S rRNA, the result will be positive. This kit detects mycoplasma contamination based on detecting the metabolites produced by the mycoplasma, so this kit will only detect the viable mycoplasma.
PCR method detects the existence of mycoplasma via amplification of 16S rRNA sequences. The mycoplasma species can be detected by PCR reactions closely related with the primer sequence. This rationale explains how the PCR methods can only detect a limited variety of mycoplasma. This kit detects mycoplasma contamination based on metabolites produced by many types of mycoplasma. When the primers used in the PCR method cannot amplify the target sequence, the result of PCR method is negative, even if there is viable mycoplasma in the sample.
The cell culture medium contains serum. During the serum preparation process, metabolites of mycoplasma maybe still retained, even if the mycoplasma has been removed. Therefore, when using this mycoplasma detection kit, it's highly recommend to use the same cell culture medium as a negative control, with the same batch of cell culture medium as the best negative control.
Yes. The metabolites produced by the mycoplasma are stable when stored at 4°C. This kit can be used to reliably detect metabolites found in the cell supernatant stored at 4°C for up to 5 days post-collection.
Limitations of This Method
Trouble Shooting
Fig A Fig B Fig C
Problem Suggestion
Problem Suggestion
Problem Suggestion
During the process of adding sample, if the pipette tip touched the filter paper of the test well, will it
influence the test result?
Can this mycoplasma detection kit be used to detect whether a cell suspension is contaminated
by mycoplasma?
No.
Yes.
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