550　　医薬品医療機器レギュラトリーサイエンス　 Vol. 50 No. 9（2019）

本誌掲載記事の無断転載・翻訳を禁止します．また，無断複写・複製は著作権法上の例外を除き禁止されています．

医薬品医療機器レギュラトリーサイエンスPharmaceutical and Medical Device Regulatory Science

Vol. 50， No. 9， 550～ 559（2019）投稿/原著

マイコプラズマ否定試験に用いるマイコプラズマ参照品に関する研究（第 1 報）

Mycoplasma arginini NBRC 111899 株の 核酸増幅法（NAT）への適用と維持管理に関する研究

林 克彦＊1, ＊2，渡辺 愛弓＊3，門脇 成武＊3，湯之前 雄太＊4，中川 香奈子＊1， 豊田 淑江＊1, ＊5，鈴木 俊宏＊3，清水 則夫＊4，工藤 由起子＊1，菊池 裕＊1, ＃

（受付：平成 31 年 4 月 12 日，受理：令和元年 7 月 10 日）

Studies on Reference Strains for Mycoplasma Testing （Part Ⅰ） Maintenance and NAT-based Detection of Mycoplasma arginini NBRC 111899

Katsuhiko HAYASHI＊1, ＊2，Ayumi WATANABE＊3，Narumu KADOWAKI＊3，

Yuta YUNOMAE＊4，Kanako NAKAGAWA＊1，Toshie KANAYASU-TOYODA＊1, ＊5，

Toshihiro SUZUKI＊3，Norio SHIMIZU＊4，Yukiko HARA-KUDO＊1 and Yutaka KIKUCHI＊1,＃

Summary　Bacteria of the Mollicutes class, which are among the smallest self-replicating forms of life, are notorious for contaminating cell cultures. Mycoplasma and Acholeplasma are especially problematic in cellular experiments and in biological medical productions such as antibody drugs. The Japanese Pharmacopoeia 17th Edition （JP17） requires Mycoplasma testing for Cell Substrates used for the Production of Biotechnological/Biological Products in the General Information. The nucleic acid amplification test （NAT） is useful for rapid detection and for isolation from small samples if the detection limit of NAT is adequately validated with two gold standards, the Culture Method or the Indicator Cell Culture Method. Mycoplasma arginini NBRC 111899 is not currently listed among the reference strains of NAT in JP17, and so here we examined subculture methods and NAT for this strain. We prepared F2, F3 and F4 subcultures of M. arginini NBRC 111899 by cultivation for approximately 42 hours and assessed growth in terms of the absorbance at 560 nm derived from phenol-red added to the culture medium. The ratios of genome copies （GC） per colony-forming unit （CFU） were between 1.79 and 5.25. These low GC/CFU values indicated that the F2, F3 and F4 subcultures are of sufficiently good quality for use as NAT references. NAT was operated with MycoTOOL PCR and a QC Sample Preparation Kit. MycoTOOL PCR detected M. arginini NBRC 111899 in 8/8, 23/24 and 1/8 CHO DG44 cell samples in a volume of 1 mL, spiked at 100, 10, 1 CFU/mL, respectively. Statistical PROBIT regression gave a limit of detection （LOD） of 9.33 CFU/mL, which is below the required detection limit for gold standards. These findings validate M. arginini NBRC 111899 as a reference strain for NAT Mycoplasma Testing in the Japanese Pharmacopoeia.

Key words　Mycoplasma , Mycoplasma arginini , Mycoplasma testing, Reference strain, Nucleic acid amplification test, NAT, PCR, Validation, Biological production