建立人類巨細胞病毒(HCMV)核酸國家標準品   全文下載

Establish the National Standard of Human Cytomegalovirus for Nucleic Acid Amplification Techniques (NAAT)

本研究參考世界衛生組織（WHO）之HCMV國際標準品製備流程，以細胞培養方式製備病毒原液，經加熱去活化，以Tris-HSA作為稀釋液，將病毒原液稀釋至適當濃度後透過體外診斷（IVD）檢測套組與In-house digital PCR兩套系統進行定量，完成候選國家與工作標準品各1,188管與1,207管。為使標準品具客觀性與公信力，邀集國內外5家實驗室參與共同標定研究，經統計分析顯示HCMV國家與工作標準品定量之濃度分別為7.7×106 IU/mL（6.89 log IU/mL）與8.7×103 IU/mL（3.94 log IU/mL）。並進行24°C、4°C、-20°C以及-80°C等4個溫度點至12個月之安定性評估，結果顯示於-20°C與-80°C儲存溫度下之濃度並無顯著變化。該標準品除供衛生福利部食品藥物管理署（下稱食藥署）作為細胞治療產品之查驗登記與品質監測及相關病毒核酸標準檢測體系用，亦可供業界與捐血中心作為分子診斷試劑與細胞治療產品研發及／或檢驗之品質管控，增加國內取得標準品之管道，協助我國再生醫療產業發展。

The preparation of the 1st national standard for human cytomegalovirus (HCMV) DNA followed the WHO international standard for HCMV and related literature. HCMV virus stock was prepared by cell culture. After heat inactivation, the inactivated virus stock was diluted with tris-HSA to appropriate concentrations and filled into 1,188 vials of candidate national standard and 1,207 vials of working reagent. Quantification was carried out by a commercial IVD kit and an in-house digital PCR assay. In order to ensure the objectivity and credibility of the 1st HCMV DNA National Standard, we invited five clinical and official control laboratories to participate in an international collaborative study. The laboratories were requested to test the candidate standards and the WHO HCMV International Standard (NIBSC code 09/162) using their routine method in three independent experiments. All data sets were calculated in International Units/mL (IU/mL). The mean contents of the candidate national standard and working reagent, calibrated against the 1st WHO HCMV International Standard were 7.7 × 106 and 8.7 × 103 IU/mL, respectively. We have also conducted the stability study for 12 months at 4 different temperatures (24°C, 4°C, -20°C and -80°C). The results indicated that the concentration of standards had no significant degradation storing at -20°C and -80°C for 12 months. Further studies on long-term stability are ongoing. The established viral national standards are not only used for registration and quality management for cellular therapy products and related viral nucleic acid standard testing systems, but also used by the industry and blood centers for the development of molecular diagnostic reagents and cellular therapy products and/or quality control of testing. We hope to enhance domestic access to standard products and assist the development of regenerative medical industry in Taiwan.