A7 - PCR: Application of nested PCR to detection of mycoplasmas

Abstract

This chapter describes the application of nested polymerase chain reaction (PCR) to detection of mycoplasmas. In a typical protocol for the nested PCR, a first-round PCR is performed with a pair of outer primers. The nested PCR is currently the most sensitive means of detecting the mycoplasmas in cell cultures or in clinical materials because a single copy of target sequence can be detected. The second-round PCR serves to confirm the specificity of the first-round PCR. The nested primer pairs recommended in this chapter consist of sequences of the 16S-23S ribosomal RNA intergenic spacer regions. High-titerd mycoplasma cultures inhibit PCR because intracellular mycoplasmal components inhibit the activity of Taq DNA polymerase. Although nested PCR may increase the risk of cross-contamination with product DNA because of its high sensitivity, it does provide a faster means of detecting low-titer infections. Materials required for the isolation of DNA from mycoplasma-contaminated biological materials are illustrated in the chapter.