目的 建立检测异嗜性小鼠白血病病毒（xenotropic murine leukemia virus，X-MuLV）的实时荧光定量反转录PCR（real time fluorescent quantitative reverse transcription PCR，RT-qPCR）方法，并进行验证及初步应用。方法 根据美国典型培养物保藏中心提供的X-MuLV基因组序列，设计特异性引物并构建包含靶序列的质粒和RNA片段；使用数字PCR定量并经连续稀释后作为标准品，建立RT-qPCR检测方法。对建立的方法进行灵敏度、特异性、准确度、精密度和耐用性验证。应用建立的方法对纳米膜过滤及阳离子层析工艺去除逆转录病毒的能力进行评估，并与致细胞病变法的评估结果进行比较。结果 建立定量检测X-MuLV的RT-qPCR方法，该方法在2.5×10¹~2.5×10⁸拷贝/μL范围内线性良好，扩增效率为98%；检测灵敏度为2.5×10¹拷贝/μL；与实验室中其他常用病毒不发生交叉反应；重复性和中间精密度验证的变异系数均不高于1%；蛋白添加及缓冲液组分对样品中病毒检测结果不产生基质效应。应用该法检测纳米膜过滤工艺中指示病毒X-MuLV的去除率，处理前后病毒下降量≥4 lg 拷贝，可认定纳滤法能够有效清除病毒，结论与致细胞病变法检测结论一致。应用于复合式阳离子交换层析时RT-qPCR方法检测结果可反映病毒清除过程中病毒颗粒分布情况。结论 建立的RT-qPCR方法检测X-MuLV灵敏度、特异性、准确度、精密度和耐用性良好，适用于生物制品生产工艺的病毒清除验证工作。

Objective To establish, verify and preliminarily apply a real time fluorescent quantitative reverse transcription PCR (RT-qPCR) detection method for xenotropic murine leukemia virus (X-MuLV). Methods Specific primers were designed according to gene sequence of X-MuLV provided by American Type Culture Collection. Recombinant plasmid and RNA standard containing target sequence were constructed, quantified through digital PCR and serially diluted to establish RT-qPCR method. Sensitivity, specificity, accuracy, precision, and robustness of RT-qPCR were investigated. The method was applied to validate virus clearance procedure by nanofiltration and chromatography, and compared with cytopathic effect assay.Results The established RT-qPCR detection method for X-MuLV showed a linear range of 2.5×10¹-2.5×10⁸ copies/μL，and the amplification efficiency was 98%. The sensitivity of detection was 2.5×10¹ copies /μL. No cross-reaction with other commonly used viruses in the lab was detected. The coefficients of variation of repeatability and intermediate precision validation were all less than 1%. Protein and buffer had no matrix effect on virus detection of samples. When the method was employed to access the X-MuLV clearance of nanofiltration process, >4 lg copies reduction was shown in virus load between pre- and post-treatment, consistent with the result of cytopathic effect assay. When applied to evaluate composite cation-exchange chromatography, the RT-qPCR method detection results reflected the distribution of X-MuLV particles throughout the process.Conclusion The RT-qPCR method established for X-MuLV has good sensitivity, specificity, accuracy, precision and robustness, applicable for virus clearance processes validation of biological products.