Mycoplasma Detection Kit (Luminescence)
Based on 1 publication(s) in Google Scholar
MCE Mycoplasma Detection Kit (Luminescence) utilizes chemiluminescence technology to detect the activity of specific enzymes in the sample, determining the presence of mycoplasma contamination.
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Storage :
-20°C, 1 year. Keep away from light and avoid repeated freeze-thaw cycles. Shipping with dry ice.
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Description & Advantages
Mycoplasma are small, structurally simple bacteria which lack a cell wall and represent one of the most prevalent and serious sources of cell line contamination. Mycoplasma contamination is invisible, as it causes no discernible change in turbidity or pH even at densities as high as 108 cells/mL, making it particularly insidious. Mycoplasma infections are highly toxic and can induce a range of changes in cell cultures, including reduced growth rates, abnormal morphology, chromosomal abnormalities, and metabolic disturbances. These changes can lead to slowed cell growth, aberrant differentiation, or even cell death, severely disrupting normal cell functions. Mycoplasma contamination not only significantly affects the reliability, repeatability, and consistency of experimental results but can also lead to experimental failure. Therefore, timely and accurate detection of mycoplasma contamination is crucial for ensuring the quality of cell cultures and the accuracy of experimental outcomes.
MCE Mycoplasma Detection Kit (Luminescence) utilizes chemiluminescence technology to detect the activity of specific enzymes in the sample, determining the presence of mycoplasma contamination. The detection principle is as follows: under the action of luciferase, ATP reacts with luciferin to produce fluorescence. Specific enzymes released by mycoplasma can catalyze the conversion of ADP to ATP. The generated ATP further reacts with luciferase, enhancing the fluorescence signal. By detecting changes in bioluminescent signals, the presence of mycoplasma contamination in the sample can be determined.
Features of MCE Mycoplasma Detection Kit (Luminescence)
1. High Sensitivity: Detection sensitivity is < 25 CFU/mL.
2. Rapid: The detection process can be completed within 30 min.
3. Accurate: Detects biologically active mycoplasma, providing more accurate results compared to the PCR assay.
Protocol
General Protocol
1. Preparation of detection solutions
1) Detection Reagent: Add 700 μL Mycoplasma-Free Water to one vial of Detection Reagent and dissolve completely. Keep at 4°C before use.
2) Detection Substrate: Add 700 μL Mycoplasma-Free Water to one vial of Detection Substrate and dissolve completely. Keep at 4°C before use.
Note: Fresh preparation is recommended. Reconstituted Detection Reagent and Detection Substrate may be stored for approximately 1 week at 4°C. For longer storage, aliquot and store for approximately 1 month at -20°C or 6 months at -80°C. Avoid repeated freeze-thaw cycles.
2. Sample preparation
When cell confluence exceeds approximately 80%, collect culture supernatant, centrifuge at 400 × g for 3 min, and use the supernatant as the test sample. Samples may be stored at 4°C and should preferably be tested within one week. Avoid repeated freeze-thaw cycles.
3. Luminescence assay
1) Equilibrate Detection Reagent, Detection Substrate, Positive Control, and samples to room temperature. Protect reagents from light whenever possible.
2) Add 50 μL sample, Positive Control, or negative control to individual wells of a white 96-well plate. Mycoplasma-Free Water, PBS, or fresh culture medium may be used as the negative control.
3) Add 50 μL Detection Reagent to each well, mix gently, incubate at room temperature for 5-10 min, and record the first luminescence value A.
4) Add 50 μL Detection Substrate to each well, mix gently while minimizing bubbles, incubate at room temperature for 10-15 min, and record the second luminescence value B.
5) Calculate the B/A ratio.
4. Interpretation
The Positive Control should give B/A ≥ 5. If B/A is below 5, reagent activity may have decreased and the assay should be repeated with fresh reagent.
| B/A ratio | Interpretation |
|---|---|
| ≤ 0.8 | Negative |
| 0.8-1.0 | Borderline; culture for another 24-48 h and retest |
| ≥ 1.0 | Positive |
Publications
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Journal Impact Factor
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Storage
-20°C, 1 year.
Keep away from light and avoid repeated freeze-thaw cycles.
Shipping with dry ice.
Components
| Components | HY-K0551-25 rxns | HY-K0551-50 rxns |
|---|---|---|
| Detection Reagent (Lyophilized) | 2 vials | 4 vials |
| Detection Substrate (Lyophilized) | 2 vials | 4 vials |
| Positive Control | 250 μL | 500 μL |
| Mycoplasma-Free Water | 1.5 mL × 2 | 1.5 mL × 4 |
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