Padronização e aplicação da PCR para detecção de contaminantes em cultivos celulares, soros e tripsinas

Abstract

The aim of this study was the standardization of PCR and the evaluation of their use to detect contaminants in cell cultures, sera and trypsin. A total of six PCR reactions were developed for detection of Mycoplasma (Mollicutes), PPV, PCV1, BLV and BVDV (two assays). Two PCR assays were also standardized, for GAPDH and -actin genes, to evaluate the efficiency of genetic material extraction. During standardization the specificity of primers, the analytical sensitivity and the analytical specificity were evaluated by amplicon restriction analyses and sequencing. After standardization, the PCR were applied to 88 cell culture samples from eight laboratories, belonging to official laboratories network and to education and research laboratories. We also analyzed 10 different batchesof trypsin and 13 different batches of bovine sera, supplied by the same laboratories. Ourresults showed the occurrence of following DNA cell culture contamination: 34.1% forMycoplasma- Mollicutes; 59.1% for PPV; 35.2% for PCV1; 23.9% for BVDV and 3.4% for BLV. Inbovine sera and trypsin samples DNA of BVDV, PCV and PPV was detected. To confirm the specificity of the PCR reactions, some cell cultures samples (amplicons) were sequenced. The PCR reactions standardized in this study allowed the detection of contaminants incell cultures, sera and trypsin samples, and could be used in a routine basis in laboratories working with these materials.