- 期刊
豬內源性逆轉錄病毒即時定量PCR檢測技術之建立與應用
Establishing and Application of the Real Time Quantitative PCR for Porcine Endogenous Retrovirus
摘要
豬內源性逆轉錄病毒（porcine endogenous retrovirus，PERV）自1997年被發現可以感染人類細胞株後，即引發熱烈討論並陸續發展出不同的診斷系統。本研究中我們應用即時定量PCR（real time quantitative PCR）之技術，建立PERV快速診斷系統，並對此檢測法進行方法確效，以期能準確測量出樣品中之PERV病毒含量。經重覆測試確認此檢驗技術之靈敏度為50個病毒核酸套組（copies），病毒含量自10^2至10^7 copies均顯示高線性化之標準曲線，且其相關係數均大於0.998；另在同次與不同次試驗間的變異係數分別為0.49%-2.21%與1.46%-2.43%，顯示此檢測系統具有高精確度及再現性。此檢測方法具有高度專一性，不會與X-MuLV、E-MuLV、PRV及BVDV等病毒產生交叉反應。同時以即時定量PCR及體外感染試驗法進行PERV定量分析，結果分別為10^（8.08）virus particles/mL 及10^6 TCID（下標 50）/mL，顯示以即時定量PCR檢測較體外感染試驗（in vitro infectivity assay）之靈敏度約高100倍。此外，我們應用此檢測法進行PERV的病毒複製動力研究，發現PK15細胞在培養第6天可產生最高之病毒力價，因此適用於製備確效用之高力價PERV種毒（virus bank）。相較於傳統體外感染試驗法的費時、費力，我們所建立的PERV即時定量PCR檢測技術具有快速、便利、高靈敏度、高專一性及高精確度等特性，未來將可應用於豬隻來源生物製劑的病毒偵測及製程中所需的病毒清除效力之評估（virus clearance evaluation）。
並列摘要
Use of porcine xenografts may be associated with a risk of transmission of microorganisms. Porcine endogenous retroviruses (PERV) are of particular concern while there is no definitive report for PERV transmission in humans. For public health issues, a highly sensitive technique needs to develop for monitoring of porcine biomaterials and their recipients. We have developed a real time quantitative PCR and further validated for accuracy, precise and reproducibility. This assay directly quantified specific product of PERV by monitoring the increase of fluorescence intensity emitted during Taqman PCR reaction. The reliable detection range is within 100 to 107 copies per reaction with high reproducibility. The intra assay variation is 0.49%-2.21 % and the inter assays variation is 1.46%-2.43%. X-MuLV, E-MuLV, PRY and BVDV was elucidated to verify the specificity of this real time quantitative PCR for PERV. Furthermore, we found that real time quantitative PCR assay is approximately 100 fold more sensitivity than in vitro infectivity assay. In this paper, we have developed the fast, sensitive, and specific assay could replace in vitro infectivity assay for virus clearance evaluation of porcine source materials process.