Description Mycoplasma contamination is a common problem in cell culture. It can alter cell characteristics. For example, some mycoplasma species rapidly consume arginine in the culture medium, competing with cells for nutrients, which slows or halts cell growth. Mycoplasma can also change cellular enzyme profiles, membrane composition, and induce chromosomal abnormalities or pathological changes. These issues can seriously compromise experimental results and lead to unreliable conclusions. Mycoplasma detection methods include culture, fluorescent staining, metabolite detection, and PCR. Culture methods require more than three weeks and cannot detect mycoplasma species that do not form colonies. Fluorescent staining has low sensitivity and requires an expensive fluorescence microscope. Metabolite detection also suffers from low sensitivity, and serum used in complete culture media—although filtered to remove mycoplasma—may still retain metabolites that cause false positives, in addition to being costly. These methods are not well-suited for routine mycoplasma monitoring in laboratories. PCR-based detection requires only a standard PCR thermal cycler and electrophoresis equipment, is technically simple, time-efficient, and cost-effective, making it ideal for routine monitoring of mycoplasma contamination in cell culture. This product uses PCR-based detection. Primers are designed based on conserved sequences in mycoplasma genomes, and amplification products are analyzed by gel electrophoresis. The kit detects more than 40 mycoplasma species, including the eight species most commonly encountered in cell culture. The limit of detection is as low as 5 copies, with an accuracy of 99%. Results are available within 3 hours. A positive result yields a 280 bp band. An internal control is included in the kit to monitor for PCR-related interference; the internal control band appears at 70 bp. Components Component Volume MycoScan PCR MasterMix 2X 625μl MycoScan Positive Control 1X 25μl Operating Instructions Sample Preparation After culturing cells for 2 days (3 days yields better results), collect 1 mL of culture supernatant and centrifuge at 3000 rpm for 5 minutes to pellet cell debris. Transfer 500 μL of the supernatant to a fresh 1.5 mL microcentrifuge tube. Place the tube in a boiling water bath (100 °C) for 10 minutes to lyse mycoplasma and release DNA. Allow the sample to cool to room temperature before proceeding to the next step. PCR Reaction 1. Reaction Setup Component Volume 2× PCR Mix 12.5 μl Sample lysate from Step 1 / Positive control / Negative control 2.5 μl (sample) / 1 μl (control) Sterile water To 25 μl Note: Positive and negative controls must be included in each experiment. The positive control is provided with the kit. Fresh basal culture medium or sterile water is recommended as the negative control. Add 1 μL of the positive control. 2. Thermal Cycling Conditions Temperature Time Cycles 94°C 2 min 1 94°C 30 sec 35 55°C 15 sec 72°C 30 sec 72°C 5 min 1 3. Electrophoresis Detection Perform electrophoresis using a 1.5–2% agarose gel. Mycoplasma-positive result: Both a 280 bp band and a 70 bp band (the latter may be faint) are present. Mycoplasma-negative result: Only a faint 70 bp band is present. Precautions Samples should be culture supernatant collected from cells grown for 2–3 days to allow trace amounts of mycoplasma to be amplified during culture. To avoid false-positive or false-negative results, always include positive and negative controls in each test. To ensure detection efficiency and avoid interference from cell genomic DNA, perform sample preparation (boiling) before PCR amplification. Collected culture supernatant can be stored briefly at 4 °C and tested within one week without affecting results. However, mycoplasma DNA released after boiling is unstable at low concentrations and prone to degradation, which may cause false negatives. Therefore, samples should be tested on the same day after sample preparation. Newly introduced cell lines should be tested for mycoplasma contamination to prevent cross-contamination of clean cultures. For routine monitoring, test in-culture cells once per month, supplemented with random weekly tests, to ensure early detection. For cells undergoing mycoplasma eradication treatment, test weekly until negative results are obtained and maintained for at least one month; thereafter, return to monthly testing with random weekly checks. FAQ 1. What should I do if a faint, smeared band appears near 280 bp after electrophoresis?Answer: It is recommended to collect fresh culture supernatant after 2–3 days of normal culture and repeat the test. 2. Why do I see ladder-like bands not at 280 bp?Answer: This is likely due to interference from cell genomic DNA. Perform sample preparation (boiling) again before amplification to eliminate genomic DNA interference. 3. Why is there no internal control band?Answer: The amount of dNTPs in the PCR system is fixed. When mycoplasma DNA in the reaction is excessive, the amplification efficiency of the internal control may decrease, and the internal control band may be absent after electrophoresis. 4. I see black granular material under the microscope, but the PCR result is negative. Why?Answer: There is no consensus on the identity of black granular material. It may be referred to as “black specks,” cell debris, inorganic particles, or fungi. Therefore, the presence of such material does not necessarily indicate mycoplasma contamination. 5. Why do results from this PCR kit differ from those obtained with other detection kits?Answer: Different kits are based on different detection principles. For example, some kits detect mycoplasma-specific metabolites. Although serum used in complete culture media is filtered to remove mycoplasma, residual metabolites may still cause false positives, leading to inconsistent results. Documents Manual
