( le was diluted with RPMI media suppleme...)A New PCR-based Mycoplasma Detection Method

le was diluted with RPMI media supplemented with 10% fetal calf serum, L-glutamine, and antibiotics. All samples were heated to 99C for 5 minutes, treated with StrataClean resin (included in kit) to remove potentially inhibitory components, and then added to the PCR mixture. Each PCR received the equivalent of 100,000 colony-forming units of the target mycoplasma/acholeplasma. This amount of mycoplasma template is similar to levels present in typical cell culture infections.

Figure 1

As demonstrated in Figure 1, all four mycoplasma samples yielded a PCR product of the expected size, while the single acholeplasma sample produced a characteristic doublet. The media control yielded no PCR product.

The mycoplasma PCR primers generated information about the species present in the culture as follows: The PCR products from a standard amplification reaction were subjected to restriction analysis with Sau 3A and analyzed using agarose gel electrophoresis. The restriction patterns for each mycoplasma-derived PCR product were unique (Figure 1); hence, individual species could now be distinguished. These data show that the kits primers amplify the target gene from mycoplasmas and their close relative, the acholeplasma. Furthermore, the PCR products can be used to obtain information about the mycoplasma/acholeplasma species present in a sample.

PCR Primers Evaluated

We subsequently evaluated the performance of the primers with bona fide laboratory samples. The experiments were conducted blind, using 12 coded cell-free supernatants provided by the Cell Culture Testing Facility at the University of California at San Francisco (UCSF). The supernatants were prepared for PCR using the s
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