( ame procedure described above. PCR reac...)A New PCR-based Mycoplasma Detection Method

ame procedure described above. PCR reactions were conducted either with or without a limiting amount of an internal control template (provided in the kit). When the internal control template was amplified, it generated a 1-kB PCR product that was easily resolved from any sample amplification product using agarose electrophoresis for analysis.

Figure 2

In the absence of the internal control template (Figure 2), we scored four samples as positive and eight samples as negative for infection. In the presence of the internal control template (Figure 3), we yielded the same results but demonstrated that one of the eight negative samples inhibited PCR amplification. For three of the contaminated samples, the PCR-amplified internal control was not inhibited to any degree, denoting a low infectious load. In the remaining infected sample, the amplified 874-bp PCR product interfered with the amplified internal control standard; this signified that high levels of mycoplasma DNA were present.

Figure 3

The PCR products amplified from the four contaminated culture supernatants were subsequently subjected to restriction analysis as described above. We were able to match the restriction patterns with the laboratory samples to those for known mycoplasmas (Table 1). Altogether, the scoring for both infection and species completely concurred with the results obtained using UCSFs established testing procedure.

Table 1
Mycoplasma Species Determined Using Different Testing Procedures