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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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TaqMan Probe Real-Time PCR for Quantitative Detection of Mycoplasma during Manufacture of Biologics

생물의약품 제조공정에서 마이코플라스마 정량 검출을 위한 TaqMan Probe Real-Time PCR

  • Lee, Jae Il (Department of Biological Sciences and Biotechnology, Hannam University) ;
  • Kim, In Seop (Department of Biological Sciences and Biotechnology, Hannam University)
  • 이재일 (한남대학교 생명.나노과학대학 생명시스템과학과) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명시스템과학과)
  • Received : 2014.07.10
  • Accepted : 2014.10.16
  • Published : 2014.10.30
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Abstract

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

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