title: Performance of PCR-based and Bioluminescent assays for mycoplasma detection.
abstract: 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.
authors: 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. Electronic address: prifalagan@gmail.com.
authors: Priscila
authors: P
authors: Falagan-Lotsch
authors: 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. Electronic address: talirialopes@gmail.com.
authors: Talíria Silva
authors: TS
authors: Lopes
authors: 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. Electronic address: cellbank@hucff.ufrj.br.
authors: Nívea
authors: N
authors: Ferreira
authors: 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. Electronic address: balthazar.nm@gmail.com.
authors: Nathália
authors: N
authors: Balthazar
authors: 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. Electronic address: cellbank@hucff.ufrj.br.
authors: Antônio M
authors: AM
authors: Monteiro
authors: 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. Electronic address: rrborojevic@gmail.com.
authors: Radovan
authors: R
authors: Borojevic
authors: 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. Electronic address: jmgranjeiro@inmetro.gov.br.
authors: José Mauro
authors: JM
authors: Granjeiro
mesh_terms: D018929:Cell Culture Techniques / Q000592:standards*; D008163:Luminescent Measurements / Q000379:methods*; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: Bioluminescent assay; Commercial kits; Mycoplasma; PCR