长链核酸扩增技术评价病毒灭活效果的研究

发布时间：2018-07-04 09:16

  本文选题：长链PCR + 病毒灭活　； 参考：《中国人民解放军军事医学科学院》2006年硕士论文

【摘要】：目的：HBV、HCV和HIV等经血传播的病毒对人类危害极大，我国仅HBV感染者就超过1.2亿。由于病毒感染窗口期的存在、新的病毒株不断出现以及检测技术还不完善等原因，通过输血或血液制品传播病毒性疾病的现象时有发生。为提高血液的安全性，，有必要对其进行病毒灭活处理。在血液病毒灭活研究工作中，常用细胞感染法评价病毒的灭活效果。该法直接、客观，能够真实反映病毒失活的程度。但由于HBV和HCV等一些病毒目前还无法在体外培养复制，病毒灭活效果难以用细胞感染法进行直观评价，真实反映这些病毒的灭活效果仍是一个难题。本研究目的是探讨长链核酸扩增技术用于评价病毒灭活效果的可行性，探索病毒灭活评价的新方法。 方法：以pTAL-Seap质粒作为模型，通过特异性酶切(BSPH-Ⅰ)及非特异性的~(60)Co-γ射线辐照(30kGy)损伤后行不同长度核酸片段(0.3kb、0.8kb、1.7kb、2.7kb、4.8kb)的PCR扩增，探讨长链核酸扩增技术评价DNA损伤的可行性。以具有体外致细胞病变效应(CPE)的伪狂犬病毒(Pseudorabie virus，PRV)作为试验病毒。PRV大量复制后经~(60)Co-γ射线辐照、低pH值(4.0±0.1)处理、巴氏法消毒(60±1.0℃)、有机溶剂／洗涤剂法(S／D法，0.3％TNBP+1％TWEEN-80)处理以及亚甲蓝／光化学法(MB／L，1.0uM亚甲蓝，40000Lux光照)灭活，提取
[Abstract]:Objective\ Because of the existence of virus infection window period, the emergence of new virus strains and the imperfect detection techniques, the phenomenon of transmission of viral diseases through blood transfusion or blood products occurs from time to time. In order to improve the safety of blood, it is necessary to inactivate the virus. In the research of blood virus inactivation, the inactivation effect of virus was evaluated by cell infection method. The method is direct, objective and can reflect the degree of virus inactivation. However, some viruses such as HBV and HCV can not be cultured and duplicated in vitro, so it is difficult to evaluate the inactivation effect by cell infection. It is still a difficult problem to truly reflect the inactivation effect of these viruses. The purpose of this study was to explore the feasibility of long chain nucleic acid amplification (LSTNA) in evaluating the inactivation effect of viruses and to explore a new method for evaluating virus inactivation. Methods: pTAL-Seap plasmid was used as a model. The DNA damage was detected by PCR amplification of different length nucleic acid fragments (0.3 kb ~ (0.8) kb ~ (2. 7) kb ~ (4) kb) after specific enzyme digestion (BSPH- 鈪

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