牛源材料中BVDV检测方法的验证及应用

Validation and application in detection method for BVDV from the bovine materials

目的对生物制品生产过程中使用的牛源材料(牛血清、牛源细胞等)进行牛病毒性腹泻病毒(BVDV)检测以保证制品的生物安全性。方法参照制药质量体系Q10(ICH Q10)对杂质的定性检测要求,采用一步法RT-PCR检测BVDV的5'-UTR保守区,并对其特异性、敏感性和重复性进行验证,然后将其应用于牛源材料中BVDV的检测。结果扩增产物为250 bp的目的片段,测序后经序列比对分析软件BLAST比对,同源性达99%,且与其他5种牛源病毒均无相关性;敏感度达1CCID50/m L。应用该方法对牛血清、牛鼻甲骨细胞(BT)、牛肾原代细胞、MDBK细胞等样品进行检测,15份为阳性,阳性检出率为12%。结论一步法RT-PCR检测BVDV,特异性强、重复性好、敏感性高,可用于牛源材料中BVDV的快速检测。

Objective To ensure the safety of biological products,we detected bovine viral diarrhea virus from the bovine materials,such as bovine serum,bovine cell,in the preparation process. Methods The one-step RT-PCR method was carried out in detection of conservative region on 5 ＇-UTR of bovine viral diarrhea virus,based on qualitative detection requirements for the impurity from ICH Q10,and its specificity,sensitivity and repeatability were validated,finally the method was applied in detection of BVDV from the bovine materials. Results A 250 bp fragment was amplified from the target,BLAST camparison was performed after sequencing and with a 99% homogenecity（ for the target）,and the fragment had no correlation with the other five species of viruses. The detection limit of this method was 1CCID50/ m L of BVDV. Different batches of bovine serum and different bovine cells as BT,MDBK and primary calf kidney cell were detected,15 samples were positive,with a positive rate 12%. Conclusion The detection method could be used as a rapid test for BVDV from bovine materials in this verification study,which has a high specificity,sensitivity and good repeatability.