Abstract

PUERTA, Juan D.; USME-CIRO, José A  and  GALLEGO-GOMEZ, Juan C. Implementation of an internal control in molecular detection of the main mycoplasma species contaminating cell cultures. Salud, Barranquilla [online]. 2013, vol.29, n.2, pp.160-173. ISSN 0120-5552.

Objective: to construct and implement an internal control for molecular detection of mycoplasma in cell cultures. Materials and Methods: Sequences of 16S ribosomal RNA of the major species of mycoplasma reported as contaminants in cell cultures were aligned, and oligonucleotides for detection were designed. For the construction of the internal control, a spacer sequence as well as sequences for hybridization of the oligonucleotides used for detection, were amplified. Amplification from the internal control and positive samples generated fragments with different sizes. Subsequently, to evaluate the effect of the internal control on the amplification of a positive sample, the assay was performed on serial dilutions of the sample in the presence and absence of the internal control. Finally, the protocol was tested using culture supernatants from different laboratories. Results: High homogeneity was observed when Mycoplasma and Acholeplasma genera were compared; however, degenerated oligonucleotides were designed to increase the efficiency of detection in all the analyzed species. It was shown that implementation of the internal control does not affect the sensitivity of detection. The results obtained with cell cultures samples highlighted the importance of the internal control. Conclusions: The use of an internal control facilitates the detection of false negative results generated by the presence of inhibitors in the sample. Implementation of the test as a quality control will benefit laboratories using cell cultures, making the results and / or products obtained more reliable.

Keywords : Molecular Detection; PCR; Internal Control; Mycoplasma; Contamination; Cell Cultures.

        · abstract in Spanish     · text in Spanish     · Spanish ( pdf )