Performance of PCR-based and Bioluminescent assays for mycoplasma detection Priscila Falagan-Lotsch a, ⁎, Talíria Silva Lopes a , Nívea Ferreira b , Nathália Balthazar a , Antônio M. Monteiro b , Radovan Borojevic b , José Mauro Granjeiro a a Laboratory of Biotechnology, Bioengineering Group, National Institute of Metrology, Quality and Technology (INMETRO), Av Nossa Senhora das Graças, n°50, Vila Operária-Xerém, Duque de Caxias, Rio de Janeiro CEP25250-020, Brazil b Rio de Janeiro Cell Bank, National Institute of Metrology, Quality and Technology (INMETRO), Brazil Av Nossa Senhora das Graças, n°50, Vila Operária-Xerém, Duque de Caxias, Rio de Janeiro CEP25250-020, Brazil abstract article info Article history: Received 27 July 2014 Received in revised form 7 July 2015 Accepted 12 August 2015 Available online 18 August 2015 Keywords: Mycoplasma PCR Bioluminescent assay Commercial kits Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Contamination of cell cultures by mycoplasma remains a significant problem in many laboratories. According to published reports, myco- plasma is present in about 5–35% of all cell cultures (Hay et al., 1989). However, the actual rates are probably higher in a significant number of laboratories that do not test for such phenomena. As reported by Rivera et al. (2009), the level of mycoplasma contamination of the cell cultures evaluated in Mexico was 88.7%. The massive presence of mycoplasma is unfortunately a big issue, especially because an array of physiological and biochemical parame- ters are affected by the presence of mycoplasmas in cell culture. It is known that mycoplasma affects cell function, growth, metabolism, mor- phology, attachment, and membrane properties, contributes to virus propagation in the cell culture, and induces chromosomal abnormalities and DNA damage, as well as cytopathic effects including plaque forma- tion (Lincoln and Gabridge, 1998). The use of contaminated eukaryotic cells may thus cause disastrous effects, since they can alter many cellular parameters, leading to unreliable experimental results and po- tentially unsafe biological products such as biopharmaceutical products used in cell therapy, tissue engineering and vaccine manufacturing (FDA, 2010). Moreover, mycoplasma contamination is a serious concern for both autologous (Gong et al., 2012) and heterologous (Albon et al., 2013) cell-based therapies and financially, represents considerable economic impact: a study estimated that approximately $350 million Journal of Microbiological Methods 118 (2015) 31–36 ⁎ Corresponding author at: INMETRO, Av Nossa Senhora das Graças, n°50, Vila Operária- Xerém, Duque de Caxias, Rio de Janeiro CEP25250-020, Brazil. E-mail addresses: prifalagan@gmail.com, pflotsch@inmetro.gov.br (P. Falagan-Lotsch), talirialopes@gmail.com (T.S. Lopes), cellbank@hucff.ufrj.br (N. Ferreira), balthazar.nm@gmail.com (N. Balthazar), cellbank@hucff.ufrj.br (A.M. Monteiro), rrborojevic@gmail.com (R. Borojevic), jmgranjeiro@inmetro.gov.br (J.M. Granjeiro). http://dx.doi.org/10.1016/j.mimet.2015.08.010 0167-7012/© 2015 Elsevier B.V. All rights reserved. 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