核黄素光化学法灭活血小板病毒方法的建立及对灭活效果的评价  

Virus Inactivation of Apheresis Platelet Concentrates Using Riboflavin and Photochemical Method:Establishment of Experimental Conditions

作　　者：许伟[1] 卫玉芝[1] 李燕萍[1] 钟涛[1] 张循善[1] 

机构地区：[1]安徽医科大学第一附属医院输血科,安徽合肥230022

出　　处：《中华医院感染学杂志》2010年第19期2910-2913,共4页Chinese Journal of Nosocomiology

摘　　要：目的建立核黄素光化学法灭活血小板病毒的试验方法,探讨PCR用于评价病毒灭活效果的可行性,观察PCR和细胞感染实验在评价核黄素光化学法对血小板病毒灭活效果的一致性。方法以人巨细胞病毒(HCMV)AD169株作为模型病毒,含不同浓度核黄素的血小板病毒悬液分别以不同辐照剂量的紫外线照射,以病毒细胞感染实验评价灭活效果,选择最佳灭活条件;提取最佳灭活条件处理后血小板悬液中的病毒核酸,以此作为PCR扩增的模板,与病毒细胞感染实验相比较。结果浓度为150μmol/L的核黄素结合辐照剂量为1500mJ/cm2紫外线照射为最佳灭活条件;当核黄素工作浓度为150μmol/L、紫外线辐照剂量<1500mJ/cm2时,2条不同长度的HCMV核酸片段均能被扩增出来,而平行的细胞感染实验证明,处理后的HCMV仍具有感染性;当核黄素工作浓度为150μmol/L、紫外线辐照剂量≥1500mJ/cm2时,547bp片段在各处理组中扩增仍为阳性,但1505bp片段扩增为阴性,平行的细胞感染实验证明了所试病毒被完全灭活。结论影响核黄素光化学法灭活病原体效果的主要因素为核黄素工作浓度及紫外线辐照剂量;PCR和病毒细胞感染试验一样能够反映出某些灭活病毒方法的效果。OBJECTIVE To establish a method that can inactivate virus using riboflavin plus ultraviolet（UV） radiation,the new technique of PCR was investigated for its feasibility in the estimation of virus inactivation and for its consistency of cell-based infection assays in estimation of virus inactivation effect.METHODS Using human cytomegalovirus（HCMV） AD169 as a model virus,adding various concentrations of riboflavin to HCMV in polyvinyl chloride（PVC） platelet conservation bag and then the mixture was exposed to different UV dosage,and the virus titer was measured by cell-based infection assays and selected the optimal conditions for inactivation.Extracted the virus nucleic acid in platelet after inactivation with the best conditions,as a template for PCR amplification,to compare with cell-based infection assays.RESULTS The riboflavin concentration of 150 μmol/L and UV intensity of 1500 mJ/cm2 were the best conditions.After HCMV was treated by riboflavin and UV,the PCR showed that the amplification of 1505 bp DNA fragment was negative and the short DNA fragment of 547 bp was amplified positively.The amplification results by PCR were well corresponding to the results of cell-based infection assays.CONCLUSIONS The main factors of virus inactivation by riboflavin plus UV are the concentration of riboflavin and the intensity of UV.The PCR technique as cell-based infection assays probably is reflecting the efficacy of some methods for virus inactivation.

关 键 词：病毒灭活 细胞培养 光敏剂 核黄素 人巨细胞病毒 聚合酶链反应 

分 类 号：R453[医药卫生—治疗学]