QPCR检测人细小病毒B19方法的建立及其在病毒去除工艺验证中的应用  

Establishment of method for detecting B19 with QPCR and application in virus removal validation

作　　者：许锬 吕家成 容新宗 黄琰 郑朝共 

机构地区：[1]成都蓉生药业有限责任公司,四川成都610041

出　　处：《中国输血杂志》2014年第9期916-919,共4页Chinese Journal of Blood Transfusion

摘　　要：目的建立1种检测人细小病毒(B19)的荧光定量PCR方法,并应用于纳米膜过滤去除病毒的工艺验证,评价在血液制品生产过程中纳米膜过滤去除B19的效果。方法用上游引物(P1)5’-GGC ACC TCT CAA AAC ACT-3’和下游引物(P2)5’-C CAC TCC TTG CTG ATA CTC-3’,在95℃3min、95℃10s52℃10 s72℃10S条件下扩增40个循环,建立检测B19的QPCR方法并验证该方法的专一性、重复性和灵敏性;最后用该方法检测纳米膜过滤去除制品中B19的效果。结果该方法实验内和实验间的精密度均<5%,最低检测限为50 copies/μL;经检测纳米膜过滤可使样品中B19滴度下降3.8Logs。结论成功建立了具有良好专一性、重复性和灵敏性的荧光定量PCR方法。Objective A Quantitative PCR method was established for detecting Human Bocavirus （B19） and applied on the validation process indicating the removal of virus. Using this method and nanometer membrane filtration,the effect of vi- res removal on the productive process of blood products was evaluated. Methods Using the following primers, （ P1 ） 5＇- GGC ACC TCT CAA AAC ACT -3＇and （P2） 5＇-CCA CTC CTT GCT GAT ACT C -3＇,at 95℃ for 3 minutes,at 95℃ for 10 seconds,at 52℃ for 10 seconds, and at 72℃ for 10 seconds,40 cycles were amplified respectively in each condition. The QPCR method used to detect BI9 concentration in samples was established with the validation of its specificity, replicability and sensitivity. Last,this method was used to evaluate the effect of B19 removal in blood products with nanometer membrane filtration. Results Both intra and inter-assay of the methods have shown precision within 〈 5% in count. The lowest detectable limit was 50 copies/μL. The titers of samples treated by nanometer membrane filtration descended by 3.89logs. Conclusion A QPCR method with good specificity, replicability and sensitivity was successfully established and provides a theoretical foundation for the rapid and accurate detection of B19 and the validation of virus removal process.

关 键 词：荧光定量PCR 特异性扩增 病毒去除 纳米膜过滤 

分 类 号：R446[医药卫生—诊断学] R373[医药卫生—临床医学]