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| Product Name | Mycoplasma Luminescence Detection Kit (ATPase Method) |
| Catalog No. | RESK-0194 |
| Description | The Mycoplasma Luminescence Detection Kit (ATPase Method) is a chemiluminescence-based kit for determining whether cell cultures and related biological samples are contaminated with mycoplasma. It is suitable for low-sensitivity detection instruments (for example, mainstream microplate luminometers) and enables rapid, reliable assessment of mycoplasma contamination in cell culture systems. Mycoplasma are small prokaryotes without a cell wall; they are unaffected by beta-lactam antibiotics such as penicillin, and some species can pass through 0.22 um filters, making them difficult to eliminate by conventional means. Because of their limited metabolic capacity, mycoplasma often attach to cell surfaces and form cryptic contamination in cell cultures, altering gene expression and metabolic states and causing abnormal growth, which compromises the reliability and reproducibility of experimental results. Routine mycoplasma monitoring of cell systems is therefore essential. Bacterial, yeast or mold contamination in cell culture can usually be observed under a light microscope, but mycoplasma, being small and wall-less, generally cannot be identified directly and requires dedicated detection methods. Common approaches such as mycoplasma culture, ELISA, biochemical assays, PCR and DNA fluorescent staining are often tedious, limited in sensitivity or time-consuming. This kit detects mycoplasma contamination based on a mycoplasma-specific enzyme activity via an ATP-dependent luciferase luminescence reaction. The workflow is simple and takes only about 15 minutes in two steps: Step 1: Add Reagent A to the sample, incubate for 5 minutes and read luminescence value A, which reflects the background ATP level of the sample. Step 2: Add Reagent B, incubate for 10 minutes and read luminescence value B. If the sample contains mycoplasma, its specific enzyme converts ADP to ATP using the proprietary substrate in Reagent B, raising reading B. The B/A ratio determines the result: a ratio > 1.2 indicates mycoplasma contamination; a ratio < 0.9 is negative; a ratio of 0.9-1.2 is borderline, and continued culture for 24-48 hours followed by retesting is recommended. |
| Intended Use | Determination of mycoplasma contamination in cell cultures and related biological samples; rapid and reliable assessment of mycoplasma status in cell culture systems. |
| Principle / Technology | Detection of a mycoplasma-specific enzyme (ATPase) activity: in the presence of the proprietary substrate in Reagent B, the mycoplasma enzyme converts ADP to ATP, which is measured by an ATP-dependent luciferase luminescence reaction. This specifically detects viable mycoplasma. |
| Detection Method | Two-step bioluminescent (ATP-dependent luciferase) readout on a chemiluminescence reader, including low-sensitivity instruments: 1. Add Reagent A, incubate 5 minutes, read value A (sample background ATP level). 2. Add Reagent B, incubate 10 minutes, read value B. Calculate the B/A ratio: > 1.2 = positive for mycoplasma contamination; < 0.9 = negative; 0.9-1.2 = borderline, continue culture for 24-48 hours and retest. Total assay time is about 15 minutes. |
| Sample Type | Cell culture supernatant (used directly); also suitable for culture media, serum and other samples. |
| Performance Range / Specifications | Unique enzyme-activity detection: based on mycoplasma-specific enzyme activity via an ATP-dependent luciferase luminescence reaction, specifically recognizing viable mycoplasma. High sensitivity: performance verification shows sensitivity superior to a competitor product (positive samples diluted 16-fold are still detected). Broad species coverage: detects 44 mycoplasma species, including pharmacopoeia-required M. orale and M. pneumoniae. Fast and simple: the entire workflow takes about 15 minutes, without lengthy culture or complex nucleic acid extraction. Objective results: clear interpretation by B/A ratio (> 1.2 positive, < 0.9 negative). Flexible samples: cell culture supernatants can be used directly; culture media and serum are also suitable. Good instrument compatibility: designed for low-sensitivity chemiluminescence readers such as mainstream microplate luminometers. Strong stability: reagents are stable for 30 days at 4 °C and remain stable after 10 repeated freeze-thaw cycles. |
| Sensitivity / LOD | Positive samples diluted 16-fold are still detected (performance verification). |
| Specificity | Specifically detects viable mycoplasma based on a mycoplasma-specific enzyme activity; covers 44 mycoplasma species including pharmacopoeia-required M. orale and M. pneumoniae. |
| Reaction Conditions / Protocol | Step 1: add Reagent A to the sample and incubate for 5 minutes; read luminescence value A (background ATP). Step 2: add Reagent B and incubate for 10 minutes; read luminescence value B. Interpret by the B/A ratio: > 1.2 positive; < 0.9 negative; 0.9-1.2 borderline (continue culture 24-48 hours and retest). Total time about 15 minutes. |
| Components / Formulation | The kit contains 4 components in total, including Reagent A (light-sensitive) and Reagent B (containing the proprietary substrate). Upon receipt, verify that all 4 components are complete and store them immediately at the indicated temperature. |
| Storage Conditions | Store all components at -25 to -15 °C. Component (Reagent) A must be protected from light. |
| Shelf Life | 1 year at -25 to -15 °C. |
| Package Specifications | 20 T, 100 T |
| Quality Control | Manufactured under a certified quality management system; customer audits are supported. |
| Shipping Conditions | All components are shipped on dry ice. |
| Expiration Date / Stability | Reagents remain stable for 30 days at 4 °C and after 10 repeated freeze-thaw cycles. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Designed for low-sensitivity chemiluminescence detection instruments, such as mainstream microplate luminometers. |
| Application Notes / Precautions | A B/A ratio in the 0.9-1.2 borderline range should be resolved by continuing culture for 24-48 hours and retesting. Protect Reagent A from light. Verify all 4 components upon receipt and store them at the indicated temperature immediately. |
For research use only, not for clinical use.
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