目的 研制首批逆转录酶活性测定国家标准品。方法 按《中国药典》2015年版三部相关要求,进行逆转录酶活性测定标准品的制备、检验,并使用热加速试验进行稳定性考察,以SYBR Green Ⅰ实时定量聚合酶链式反应法分别在4个实验室进行协作标定。结果 制备的逆转录酶活性测定标准品外观、无菌检查合格,水分含量为1.3%,分装精度为0.75%。在-20、4、25和37 ℃放置3个月和6个月的标准品与-70 ℃保存的标准品相比酶活性均没有下降。经4个实验室协作标定共22次,标准品酶活性的几何平均值为每支26 U,95%置信区间为每支22.95~30.45 U。结论 该批逆转录酶活性测定标准品各项指标均符合要求,可作为国家标准品使用,酶活性定为每支26 U。
Abstract
OBJECTIVE To establish the first national standard substance of reverse transcriptase for activity assays.METHODS The standard substance of reverse transcriptase was prepared and tested according to the requirements in Chinese Pharmacopoeia (Volume Ⅲ,2015 edition). The stability of the standard substance was evaluated by thermal acceleration stability test. The activity was calibrated collaboratively by four laboratories using SYBR GreenⅠreal-time quantitative PCR method. RESULTS The prepared standard substance was qualified in appearance and sterility tests. The moisture content was 1.3% and the dispensing accuracy was 0.75%. Thermal acceleration stability test showed that the enzyme activity of the standard substance stored at -20, 4, 25 and 37 ℃ for 3 and 6 months was not decreased compared with that stored at -70 ℃. Twenty-two calibration tests by four laboratories showed that the geometric mean enzyme activity of the standard was 26 U·vial-1,with a 95% confidence interval of 22.95-30.45 U·vial-1. CONCLUSION The candidate standard substance of reverse transcriptase is judged to be suitable to serve as a Chinese national standard for its activity assays, and the enzyme activity specification is established to be 26 U·vial-1.
关键词
逆转录酶 /
国家标准品 /
酶活性
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Key words
reverse transcriptase /
Chinese national standard /
enzyme activity
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中图分类号：
R927
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脚注
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基金
863计划资助项目(2012AA020805)
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