Lonza’s (www.lonza.com) MycoAlert® Mycoplasma Detection assay is a generic biochemical test for Mycoplasma and other mollicutes. It offers the possibility to test cultures for mycoplasmal contamination whenever the cells are passaged and gives the user a profile of their cells, allowing them to spot infections at an early stage (Figure 1).
The MycoAlert Assay detects the activity of mycoplasmal enzymes, which generate ATP. When provided with specific substrates, these enzymes are able to convert ADP to ATP, which is then detected using the bioluminescent luciferase/luciferin reaction. The kinetics of this reaction are illustrated in Figure 2.
The MycoAlert Assay is simple to perform. First, the MycoAlert Reagent, containing the bioluminescent detection system and a detergent that will lyse any Mycoplasma present, is added to the test sample. The sample is incubated at room temperature for five minutes, establishing an ATP baseline measurement. Then, a one-second integrated light reading is taken (Reading A in Figure 2).
Next, the MycoAlert Substrate is added. This reagent contains the specific substrates for the mycoplasmal enzymes. After a 10-minute incubation at room temperature, a second reading (Reading B in Figure 2) is taken. By dividing Reading B by Reading A, a ratio is obtained.
If Mycoplasma are present, the enzymes convert ADP to ATP, shown by a dramatic increase in the number of RLUs (red line). Hence, the value of the ratio will be above one, indicating a positive sample. If no Mycoplasma are present, the baseline ATP level will gradually drop, as ATP is consumed by the luciferase/luciferin reaction (blue line). In this case, the value of the ratio will be below one, indicating a negative sample.
The MycoAlert Assay has been optimized to work with cell culture samples under normal culture conditions. It does not require any modifications to be made to the samples and can be conducted in the presence of antibiotics and colored culture medium containing serum. To date, 39 species have been tested and detected successfully by MycoAlert (Table 1). The tool will detect mollicutes of mammalian, avian, insect, and plant origin.
Being a biochemical assay that detects the activity of specific mycoplasmal enzymes, false-positive results will not be generated by eukaryotic cells that do not possess the same enzymes. The use of a selective lysis procedure means that any prokaryotes other than mollicutes that may express the same enzymes will not be detected (Table 2).
MycoAlert was used to monitor the progression of a Mycoplasma infection. K562 (human leukaemia) cells were infected with two common species of Mycoplasma and monitored regularly by removing a small sample of the culture supernatant. The infection is apparent less than three days after infection (Figure 3).
Routine monitoring of all cell lines in culture showed a covert Mycoplasma infection that was subsequently identified as M. hyorhinis. The cell lines were definitely identified as being positive or negative for Mycoplasma by the use of the MycoAlert Assay (Figure 4). The results were verified by PCR.
MycoAlert Mycoplasma detection assay offers the opportunity for routine testing of cell cultures and has been designed to easily incorporate into standard tissue culture regimes. The assay can be completed in approximately 20 minutes and provides a definitive answer. It requires no specialist treatment of the cells prior to testing and uses spent media, which would be discarded with no need to include precious cells.