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This mycoplasma test kit is designed using the activity of a unique enzyme in mycoplasma. This enzyme can decompose the unique substrate in the mycoplasma detection reagent while converting ADP into ATP. Luciferase catalyzes the oxidation of luciferin in the presence of ATP and emitting bioluminescence, which can be measured by a luminometer to reflect the presence of mycoplasma contamination in the sample to be tested. The entire detection process is simple to operate, only two steps, and takes about 15 minutes. This method has high sensitivity and detects mycoplasma with real biological activity, so the detection result is more accurate than the PCR method.
Storage and transportation
Transport with wet ice; store at -20°C, and store Mycoplasma Detection Reagent A in the dark; the validity period is 12 months.
1. Take an appropriate amount (1 mL is enough) to culture the cell supernatant for 3-6 days and centrifuge at 400 g for 3 min to remove a small amount of floating cells or debris. Take the supernatant for immediate detection, or store at 4°C for detection within a week, or -80℃ for testing within half a year;
2. Equilibrate all test reagents and test samples to room temperature, the most suitable temperature is 20-25℃;
3. Add 50 μL of the sample to be tested and a negative control (such as sterile water or PBS) to a 96-well test plate (non-transparent plate, a special 96-well white board is recommended);
4. Add 50 μL of Mycoplasma Detection Reagent A, mix gently to avoid air bubbles, and store at room temperature (20-25°C) in the dark for 5 minutes. Then use a microplate reader with chemiluminescence detection to perform chemiluminescence detection, and the meter reads as A. (Please adjust the corresponding parameters appropriately according to the sensitivity of the instrument, the detection time of each hole is generally 0.25-1 s);
5. Add 50 μL of Mycoplasma Detection Reagent B, mix gently to avoid air bubbles, and store at room temperature (20-25°C) in the dark for 10 minutes. Then use a microplate reader with chemiluminescence detection to perform chemiluminescence detection, and the meter reads as B. (Note: Please strictly follow the test 10 min after adding Mycoplasma Detection Reagent B, and it should not be advanced or delayed, otherwise it will affect the result judgment);
6. Calculate the ratio (Ratio)=reading value B/reading value A.
A. If B/A>1.1, it means that there is mycoplasma contamination in the cell culture;
B. If B/A<0.9, it means that there is no mycoplasma contamination in the cell culture;
C. If the B/A ratio is between 0.9-1.1, it is recommended to continue culturing the cells for 24-48 h, and then test again to determine whether there is mycoplasma contamination. If the B/A ratio is still between 0.9-1.1, the cell culture is free of mycoplasma contamination and is mycoplasma negative.
1. The Mycoplasma Detection Reagent A contains luciferase, which will be gradually inactivated by repeated freezing and thawing. It is recommended that the aliquots should be properly stored after the first thawing, and the aliquot container should be clean and pollution-free.
2. It is strongly recommended to use a white or black opaque 96-well plate for testing. The use of an ordinary transparent 96-well plate will cause interference with adjacent testing wells.
3. The surface of human skin is rich in ATP. Please wear experimental gloves and masks when testing. Other consumables should also be clean and pollution-free to prevent the introduction of ATP pollution from external sources.
4. For your safety and health, please wear lab coats and disposable gloves for operation.