荧光定量PCR法评价核黄素光化学法灭活全血病毒的效果  

Evaluating virus inactivation effect of blood contaminated with human Herpesvirus 8 via riboflavin and ultraviolet treatment by qPCR

作　　者：朱绍汶[1] 林红[1] 刘衍春[1] 黄成垠[1] 孙俊[1] 

机构地区：[1]江苏省血液中心,南京210042

出　　处：《江苏预防医学》2016年第4期416-420,共5页Jiangsu Journal of Preventive Medicine

基　　金：江苏省卫生厅国际交流支撑计划(JSH-2013-019)

摘　　要：目的 了解荧光定量PCR法（qPCR）评价核黄素光化学法灭活含疱疹病毒8型（HHV-8）全血的效果。方法 在常规检测合格的全血中,分别加入含105、106、107 copy/mL HHV-8的淋巴细胞BC-3和核黄素,以40-160J/mLRBC的紫外光辐照进行病毒灭活。以HHV-8ORF26为中心设计4对PCR套式扩增引物（产物长度为2 998、1 252、561、206bp）,构建和优化PCR预扩增条件,以qPCR检测灭活前、后4个片段在不同辐照条件下的扩增产物经梯度稀释的Ct值,计算相应的病毒滴度ΔLog值,以评估对病毒核酸的损伤情况。结果 核黄素光化学法对全血中HHV-8核酸有损伤作用,PCR预扩增HV-1（2 998bp）片段可用于评估辐照后HHV-8的病毒核酸损伤情况。病毒滴度ΔLog值随着病毒滴度、辐照强度和扩增片段长度的增加,最高达1.9Log值。结论 在HHV-8高流行区缺乏常规血液筛查HHV-8方法情况下,可采用核黄素光化学法进行体外灭活,降低其传播的可能性。Objective To evaluate virus inactivation effect of blood contaminated with human Herpesvirus 8（HHV-8） via ri- boflavin and ultraviolet treatment by qPCR. Methods Qualified human whole blood was inoculated with BC-3 cells which con- tained 10^5 , 10^6 , 10^7 copy/mL HHV-8 and riboflavin, which was treated by 40-160 J/mLRIx： ultraviolet（UV） irradiation to inacti- vate virus. Four set of primers（products＇ sizes of 2 998 bp, 1 252 bp,561 bp and 206 bp）were designed based on HHV-8 ORF26 sequences. The PCR conditions were optimized. The cycle threshold （Ct） values using 4 set of primers were detected by qPCR amplification after serial dilution before and after different irradiation treatments. Log Ct were calculated to evaluate dam- age of virus DNA. Results Riboflavin and UV irradiation treatment can damage HHV-8 virus DNA in whole blood. Amplifica- tion of HV-1 fragment （2 998bp） could be used to evaluate damage of virus DNA. ALog value increased with HHV-8 virus load,irradiation dose and amplicon length, with maximum value of 1.9 Log. Conclusion Riboflavin and UV irradiation treat- ment could be applied in vivo to reduce HHV-8 transmission risk in high endemic areas lack of proper screen techniques.

关 键 词：人疱疹病毒8型 病毒灭活 PCR 核黄素光化学法 

分 类 号：R752[医药卫生—皮肤病学与性病学]