Anwaruzzaman Mohammad, Wang Wensheng, Wang Eunice, Erfe Lolita, Lee Janice, Liu Shengjiang
Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
Biologicals. 2015 Jul;43(4):256-65. doi: 10.1016/j.biologicals.2015.04.001. Epub 2015 May 18.
Infectivity and reverse transcriptase quantitative real-time polymerase chain reaction (qRT-PCR) assays have been optimized and validated for xenotropic murine leukemia virus (X-MuLV) detection. We have evaluated the assays systematically with regard to specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), and precision. Both assays are specific for X-MuLV detection, with a linear detection range of 0.6-5.6 log(10) TCID(50)/mL for the infectivity assay, and 1.4-6.5 log(10) particles/mL for the qRT-PCR assay. The LLOD and LLOQ of the infectivity and the qRT-PCR assays are determined as 0.5 and 1.0 log(10)/mL, and 1.4 and 2.2 log(10)/mL. The inter-assay repeatability of qRT-PCR assay (4.2% coefficient of variation [CV]) is higher than the infectivity assay (7.9% CV). We have shown that both assays are closely correlated (r = 0.85, P < 0.05, n = 22). The particle/infectivity ratio is determined as 66. Both assays were applied to evaluate virus removal using virus clearance samples of chromatographic and filtration processes. Here, we have demonstrated that the qRT-PCR assay is much faster in testing and is approximately 8-fold more sensitive than the infectivity assay. Therefore, the qRT-PCR assay can replace the infectivity assay in many cases, but both assays are complementary in elucidating the mechanism of virus inactivation and removal in virus clearance validation.
已对嗜异性小鼠白血病病毒（X-MuLV）检测的感染性和逆转录酶定量实时聚合酶链反应（qRT-PCR）测定法进行了优化和验证。我们已系统评估了这些测定法的特异性、线性、检测下限（LLOD）、定量下限（LLOQ）和精密度。两种测定法对X-MuLV检测均具有特异性，感染性测定法的线性检测范围为0.6-5.6 log(10) TCID(50)/mL，qRT-PCR测定法的线性检测范围为1.4-6.5 log(10)颗粒/mL。感染性测定法和qRT-PCR测定法的LLOD和LLOQ分别确定为0.5和1.0 log(10)/mL，以及1.4和2.2 log(10)/mL。qRT-PCR测定法的批间重复性（变异系数[CV]为4.2%）高于感染性测定法（CV为7.9%）。我们已表明两种测定法密切相关（r = 0.85，P < 0.05，n = 22）。颗粒/感染性比率确定为66。两种测定法均用于使用色谱和过滤过程的病毒清除样品评估病毒去除情况。在此，我们已证明qRT-PCR测定法检测速度快得多，且比感染性测定法敏感约8倍。因此，在许多情况下qRT-PCR测定法可替代感染性测定法，但在阐明病毒清除验证中病毒灭活和去除机制方面，两种测定法是互补的。
