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Highly-sensitive PCR assay designed for the simple and fast detection of mycoplasma contamination in cell cultures. Includes ready-to-use reagents to perform 20 tests, each with internal control.
The EZ-PCR™ Mycoplasma Detection Kit is a highly-sensitive PCR assay designed to test for the presence of over 90 species of Mycoplasma, Acholeplasma, and Spiroplasma in cell cultures with a detection limit of 10 CFU/mL. The kit contains a ready-to-use PCR reaction mix that includes highly-optimized mycoplasma-specific primers, dNTP mix, and Taq polymerase. Internal and positive controls are also included in order to test for false negatives and for simplistic result interpretation, respectively.
With a ready-to-use format and easy-to-follow protocol, samples can be prepared in as little as 10 minutes with accurate results seen in only a few hours, making it an ideal system for routine mycoplasma screening.
Figure 1. Gel electrophoresis results obtained following PCR reaction preparation and amplification. Eight total reactions, including six samples, one negative control, and one positive control were tested, each of which contain the internal control to rule out PCR inhibition (i.e. false negatives). As shown, Sample 4 produced a mycoplasma-positive band at 270bp in addition to the internal control band at 357bp, while Samples 1, 2, 3, 5, and 6 produced only internal control bands and are thus negative for mycoplasma. Image courtesy of WiCell Research Institute.
Figure 2. Images represent examples of mycoplasma that are capable of infecting all cell culture labs. Mycoplasma can be self-replicating and are the smallest known free-living prokaryote. Mycoplasma contamination can not be seen under standard microscopes. Regulatory guidance recommends that all products derived from mammalian cell culture be tested for the presence of mycoplasma.
Mycoplasma are one of the most common, yet elusive, contaminants of mammalian cell cultures. As the smallest known free-living organism, mycoplasma are a pervasive, parasitic species of highly-infectious bacteria that are estimated to contaminate between 15-35% of all continuous cell cultures worldwide. While other mycoplasma detection methods are available, PCR-based assays have the highest sensitivity with minimal preparation time for early detection in a rapid and simple manner, when compared to other methods.
The earlier mycoplasma contamination is discovered, the simpler it is to treat. The easy-to-use and cost-efficient EZ-PCR™ Mycoplasma Detection Kit was designed for highly-sensitive routine screening and detection of mycoplasma and other closely related species. To avoid major contamination, testing should be carried out minimally every 2 weeks to 3 months, especially when shared incubator spaces are used, as well as prior to the incorporation of new cultures from outside sources.
Efficient and precise PCR-based testing for mycoplasma infection should also be conducted throughout the cell culture manufacturing process from inoculation through harvest, involving routine tests of raw materials, cell banks, and viral seed stocks.
If cell culture media is older than 48 hours when collected, cellular byproducts can accumulate that can inhibit the PCR reaction. In this case, it is best to replace media and collect for PCR between 24 and 48 hours later. If a sample collected after the 48-hour timeframe must be used, DNA extraction is recommended prior to PCR amplification.
Running the 2% agarose gel at 100 volts for 75 minutes is a recommendation but may vary depending on the apparatus. If recommendations for specific gel electrophoresis setups are needed, one option is to prepare and run a pre-made 2% E Gel agarose gel (Thermo Fisher) according to protocol with the appropriate program on the E-Gel® iBase™ Power System (Thermo Fisher) to run for 26 minutes.
To know for sure, a sample producing a faint positive band may be re-tested. If a faint band persists in the subsequent assay, the sample should be considered mycoplasma positive. If the faint band no longer appears in the subsequent assay, the sample is negative.
Each kit has a Reaction Mix (200μl), Buffer Solution (1.0ml), Positive Template Control (20μl), Internal control DNA template (20μl), and Internal control primers mix (100μl) - enough reagents to conduct 20 tests.
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