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| Product Name | One-Step Colorimetric Mycoplasma Detection Kit |
| Catalog No. | RESK-0190 |
| Description | The One-Step Colorimetric Mycoplasma Detection Kit is a rapid detection product developed on a proprietary isothermal amplification technology for the detection of mycoplasma contamination in cell culture fluids. Compared with previous mycoplasma detection products, this upgraded kit greatly reduces the false-positive rate, improves detection accuracy, and enhances the discrimination between negative and positive results. After amplification, the reaction can be left at room temperature for a period of time without any negative-to-positive conversion, so result interpretation is not affected. The principle is that if a cell culture is contaminated with mycoplasma, conserved sequences of mycoplasma DNA are amplified rapidly and in large amounts, causing the reaction solution to change from blue-violet to sky blue. The result is visible to the naked eye, with no electrophoresis required. The kit detects a broad range of mycoplasma species, including 22 species and the 8 species most commonly found in cell culture. Traditional nested PCR-based mycoplasma detection is susceptible to inhibitors present in cell culture supernatants, producing false-negative results, and requires post-amplification opening of tubes for gel electrophoresis, which increases the risk of contamination-related false positives. This one-step kit is completely free of these drawbacks, and its detection sensitivity and accuracy are far superior to PCR-based methods. |
| Intended Use | For the rapid detection of mycoplasma contamination in cell culture supernatants. |
| Principle / Technology | Based on isothermal amplification technology. If the cell culture is contaminated with mycoplasma, conserved mycoplasma DNA sequences are amplified rapidly and in large amounts. The reaction system contains Mg2+ and HNB indicator: the initial Mg2+-HNB complex appears blue-violet; as amplification proceeds, accumulating pyrophosphate binds Mg2+, releasing free HNB and turning the solution sky blue. The result is read by the naked eye without electrophoresis. The kit also contains UDG enzyme, which eliminates aerosol contamination from dU-containing amplification products, greatly suppressing false-positive reactions and improving accuracy. The UDG enzyme works at room temperature, is temperature-sensitive, and is easily inactivated without affecting the results. |
| Detection Method | Colorimetric visual readout (naked eye): blue-violet indicates a negative result and sky blue indicates a positive result. No electrophoresis or instrument-based readout is required. |
| Sample Type | Cell culture supernatant (1 uL of supernatant suspected of mycoplasma contamination per reaction). |
| Performance Range / Specifications | Detects 22 mycoplasma species, including the 8 species commonly found in cell culture. Clear result discrimination with a well-defined reference standard, high detection rate, and strong negative/positive discrimination. In comparative testing at 63 °C with reaction times from 30 to 50 min, the kit showed far greater stability than competing products, with no false positives. Extended reaction tolerance: reaction solutions left for 60, 90, or 120 min showed no weak-positive conversion, and the no-template control (NTC) showed no obvious color difference from its original state. Post-reaction stability at room temperature: after the 63 °C reaction, no 95 °C inactivation is required; reactions can be left at room temperature for 3 days with no obvious change in negative or positive results. |
| Sensitivity / LOD | In head-to-head comparative testing using 1 uL of mycoplasma DNA as template, the kit's 30 min detection performance was comparable to that of competing products (competitor detection levels at 30 min ranged from 1000 copies/uL), while showing far higher stability and no false positives. Sensitivity and accuracy are far superior to conventional PCR-based methods. |
| Specificity | Detects 22 mycoplasma species, including the 8 species commonly encountered in cell culture. |
| Reaction Conditions / Protocol | Add 1 uL of cell culture supernatant suspected of mycoplasma contamination to the reaction system. Place the sample at 25-37 °C for 5 min, then incubate at a constant 63 °C for 60 min in a water bath or PCR instrument. Observe the color change: a shift from blue-violet to sky blue indicates a positive result, which is visible to the naked eye without electrophoresis. |
| Components / Formulation | Isothermal amplification reaction system with Mg2+ and HNB colorimetric indicator, UDG enzyme for carryover contamination control, and a positive control (linearized fragment, 10^5 copies/uL). |
| Storage Conditions | Store at -25 to -15 °C protected from light. If not used for an extended period, keep protected from light. |
| Shelf Life | 18 months. |
| Package Specifications | 25 T 100 T 5 T |
| Product Form | Liquid reaction reagent kit. |
| Quality Control | Comparative performance testing showed that at 63 °C the kit maintained stable results from 30 to 50 min with no false positives, whereas competing products showed NTC positive conversion and indistinguishable negative/positive results within the same time frame. Reactions left at room temperature for 3 days after completion showed no change in negative or positive results. The positive control is a linearized fragment at 10^5 copies/uL (not live mycoplasma). |
| Concentration | Positive control: 10^5 copies/uL (linearized DNA fragment). |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Stable for 18 months when stored at -25 to -15 °C protected from light. After the reaction is complete, results remain stable at room temperature for up to 3 days. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Only suitable for testing cell culture samples. Not suitable for organic solvents (e.g., ethanol, DMSO) or strongly acidic or alkaline environments, where the reagent is unstable and prone to discoloration. |
| Application Notes / Precautions | The kit is intended only for cell culture samples; do not use it with organic solvents (such as ethanol or DMSO) or strong acid/strong base environments, as the reagent is unstable and easily denatured under these conditions, causing the color to turn sky blue. Reagents containing ions that interact with Mg2+ (e.g., sodium dihydrogen phosphate) can trigger an immediate chemical color reaction upon sample addition. EDTA chelates calcium and magnesium ions, which can accelerate the color-change reaction; therefore samples such as trypsin-EDTA are not suitable for direct testing. After amplification, the reaction can be left at room temperature for a period without negative-to-positive conversion, so result interpretation is not affected. |
| Heat Inactivation | No 95 °C heat inactivation is required after the 63 °C reaction; the reaction can be placed directly at room temperature for up to 3 days with no obvious difference between negative and positive results. The included UDG enzyme works at room temperature and is temperature-sensitive and easily inactivated. |
For research use only, not for clinical use.
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