Abstract

BURGHER PULGARON, Yaima et al. Polymerase chain reaction with internal amplification control to detect mycoplasmas in cell cultures and raw materials. Rev Salud Anim. [online]. 2021, vol.43, n.3  Epub Dec 01, 2021. ISSN 2224-4700.

The presence of polymerase chain reaction (PCR) inhibitory substances in cell cultures and raw materials used in the manufacture of monoclonal antibodies could limit the usefulness of this method to detect mycoplasma contamination. The objective of this research was to determine the sensitivity and specificity of a PCR method to detect mycoplasma deoxyribonucleic acid (DNA) in MDCK and HeLa cell lines, hybridoma cells, ascitic fluid and cell supernatant containing monoclonal antibodies. The effect of a thermal shock-based mycoplasma DNA extraction method on PCR sensitivity, as well as the performance of two mycoplasma DNA purification methods: silica/guanidinium thiocyanate and a commercial DNA purification kit, were evaluated. In addition, an internal amplification control (IAC) was optimized to detect inhibitory samples from these matrices after thermal shock treatment. PCR-IAC was specific for the amplification of mycoplasma DNA. The inclusion of IAC plasmid at the concentration selected did not decrease the sensitivity of PCR in mycoplasma-spiked matrices. Seventy-eight samples were analyzed; all represented the matrices under study. As a result, all matrices showed PCR inhibition after thermal shock. The inhibitory effect decreased when silica/guanidinium thiocyanate or commercial DNA purification kit was used. PCR-IAC detected 65 % of inhibitory samples and revealed differential susceptibility to inhibitors among different samples of the same matrix.

Keywords : PCR; inhibition; internal amplification control; monoclonal antibodies; mycoplasma.

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