重组细胞中鼠细小病毒检测方法的建立及初步应用

目的建立生物技术产品用重组细胞中鼠细小病毒(Murine minute virus,MMV)污染的检测方法,并进行验证及初步应用。方法以NB324K细胞为指示细胞,建立MMV感染性检测方法,并以不同CCID50的MMV分别感染NB324K细胞,验证方法的灵敏度;通过设计针对编码非结构蛋白1(Non-structural protein 1,NS1)保守序列的引物和探针,建立用于重组细胞中MMV检测的实时荧光定量PCR,并对其线性、特异性、精密性、灵敏度、最低检测限及试验可行性和干扰性进行验证;将NB324K细胞感染试验与荧光定量PCR法相结合,建立NB324K细胞感染-PCR法,并通过对大量样本的检测,分析荧光定量PCR法和NB324K细胞感染-PCR法的可行性。结果 NB324K细胞感染试验的灵敏度为0.2 CCID50;荧光定量PCR检测方法最佳线性范围为1×108～1×104拷贝/μl,R2达0.99以上,特异性良好,与其他种属的细小病毒均无交叉反应,试验内和试验间Ct值的变异系数(CV)均小于5%,试验内病毒定量拷贝数的CV在20%～30%之间,试验间病毒定量拷贝数的CV在20%～50%之间,灵敏度为5×103拷贝/μl,最低感染性病毒颗粒检测限为2 CCID50。该方法能够用于细胞样品中MMV的检测,部分样品基质对病毒的检测具有一定的干扰性。NB324K细胞感染-PCR法的灵敏度为0.02 CCID50,检测时间可缩短为96 h,与荧光定量PCR法分别对47份样品进行检测,结果均为阴性。结论成功建立了MMV荧光定量PCR检测方法和NB324K细胞感染-PCR法,可应用于重组细胞中MMV污染的检测,为进一步提高生产用重组细胞的安全性奠定了基础。

Development and preliminary application of a method for detection of murine minute virus in recombinant cells

Objective To develop,verify and preliminarily apply a method for detection of murine minute virus(MMV)in recombinant cells used for biotechnological products.Methods An infectious assay on MMV was developed using NB324K cells as indicator cells.The cells were infected with MMV at various CCID50 to verify the sensitivity of the method.The primers and probes were designed according to the conserved sequence encoding non-structural protein 1(NS1),based on which a real-time fluorescent quantitative PCR for MMV in recombinant cells was developed,and verified for linearity,specificity,precision,sensitivity,minimum detection limit,feasibility and interference.A NB234K cell infection-PCR method was developed by combining NB324K cell infectivity assay with real-time fluorescent quantitative PCR.The feasibilities of NB234K cell infection-PCR and real-time fluorescent quantitative PCR methods were analyzed by detection of a large quantity of samples.Results The sensitivity of NB324K infectivity assay was 0.2 CCID50.Real-time fluorescent quantitative PCR showed a linear range of 1 × 108 ～ 1 × 104 copies / μl,a R2 value of more than 0.99,and a high specificity,while showed no cross-reactions with other minute viruses.Both the intra-and inter-CVs were less than 5% in Ct values,and 20% ～ 30% and 20% ～ 50% respectively in copies.The sensitivity of real-time fluorescent quantitative PCR was 5 × 104 copies / μl,while the minimum detection limit for infectious virus particles was 2 CCID50.The method might be used for detection of MMV in cells,while partial cell substrates showed a certain interference to the detection result.The sensitivity of NB324K cell infection-PCR was 0.02 CCID50,while the time for detection was shortened to 96 h.All the detection results of 47 samples by real-time fluorescent quantitative PCR and NB324K cell infection-PCR were negative.Conclusion The realtime fluorescent quantitative PCR and NB324K cell infection-PCR methods were developed successfully,which might be used for detection of MMV in recombinant cells.It laid a foundation of improvement of safety of recombinant cells for production.