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Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

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Abstract

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

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Acknowledgments

The authors would like to express their appreciation to Dr. Ehsan Mostafavi (Head of the Department of Epidemiology, Pasteur Institute of Iran) and also would like to thank National Cell Bank of Iran at Pasteur Institute for their financial support.

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Authors and Affiliations

  1. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

    Vahid Molla Kazemiha, Shahin Bonakdar, Amir Amanzadeh, Shahram Azari & Mohammad Ali Shokrgozar

  2. Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran

    Arash Memarnejadian

  3. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Reza Mahdian

  4. Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Shirin Shahbazi

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  1. Vahid Molla Kazemiha
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  2. Shahin Bonakdar
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  3. Amir Amanzadeh
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  4. Shahram Azari
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  5. Arash Memarnejadian
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  7. Mohammad Ali Shokrgozar
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  8. Reza Mahdian
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Correspondence to Mohammad Ali Shokrgozar or Reza Mahdian.

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Molla Kazemiha, V., Bonakdar, S., Amanzadeh, A. et al. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Cytotechnology 68, 1063–1080 (2016). https://doi.org/10.1007/s10616-015-9862-0

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