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目的优化人巨细胞病毒(human cytomegalovirus,HCMV)的培养条件,并建立一种快速检测HCMV滴度的方法。方法以人胚肺二倍体细胞(MRC-5)为病毒传代和滴度测定用细胞,对病毒接种量、培养温度、保护剂种类等条件进行优化。将本实验室建立的快速微量中和试验-感染细胞核染色法用于病毒滴度检测,并与蚀斑形成法及微量细胞病变法进行比较,采用Behrens-Karher法计算病毒滴度。结果 HCMV接种MRC-5细胞后在37℃培养可出现明显病变;MOI为1.0时,病毒滴度基本达到平台;病毒收获时加入1%人血白蛋白或10%FBS保护剂可维持病毒滴度稳定。同一病毒液经蚀斑形成法检测病毒滴度为5.707 lg PFU/ml,微量细胞病变法和感染细胞核染色法检测病毒滴度分别为5.633和5.667 lg CCID50/ml,3种方法的检测结果差异无统计学意义(P>0.05)。结论HCMV接种MRC-5的最佳培养条件为:病毒接种量1.0 MOI,在37℃培养,加入1%人血白蛋白或10%FBS作为保护剂。建立的感染细胞核染色法可用于HCMV滴度的快速测定,为抗病毒物质的开发及效果评价提供了一种新的技术手段。
Objective To optimize the culture conditions of human cytomegalovirus (HCMV) and to establish a rapid method for detecting HCMV titer. Methods Human embryonic lung diploid cells (MRC-5) were used as the passage cells for virus passage and titers. The conditions of inoculum size, culture temperature and protective agent were optimized. The rapid micro-neutralization assay established by our laboratory-infected cell nucleus staining method was used to detect the virus titer, compared with the plaque formation method and the micro-cytopathic method, and the virus titer was calculated by the Behrens-Karher method. Results MRC-5 cells inoculated with HCMV showed obvious pathological changes after inoculation at 37 ℃. When the MOI was 1.0, the virus titer basically reached the plateau. The viral titer was maintained by adding 1% human serum albumin or 10% FBS at harvest stable. The virus titer detected by plaque formation was 5.707 lg PFU / ml for the same virus solution, and 5.633 and 5.667 lg CCID50 / ml for viral cytopathology and infected nuclei staining, respectively Statistical significance (P> 0.05). CONCLUSION: The optimal culture conditions for MRC-5 inoculated with HCMV are as follows: the virus inoculation amount is 1.0 MOI, cultured at 37 ℃, and 1% human serum albumin or 10% FBS is added as the protective agent. The established method of infected nuclei staining can be used for the rapid determination of HCMV titer, which provides a new technical means for the development of anti-viral substances and the evaluation of effect.