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首页> 外文期刊>Biologicals: Journal of the International Association of Biological Standardization >Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation
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Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation

机译：评估感染性和逆转录酶实时聚合酶链反应检测用于检测清除病毒的异种鼠白血病病毒的评估

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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) TCID50/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 22 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. (C) 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
机译：感染性和逆转录酶定量实时聚合酶链反应（qRT-PCR）分析已经过优化和验证，可用于异种鼠白血病病毒（X-MuLV）检测。我们已针对特异性，线性，检测下限（LLOD），定量下限（LLOQ）和精密度系统地评估了这些检测方法。两种测定法均对X-MuLV检测具有特异性，对于传染性测定法，线性检测范围为0.6-5.6 log（10）TCID50 / mL，对于qRT-PCR测定法，线性检测范围为1.4-6.5 log（10）颗粒/ mL。传染性和qRT-PCR分析的LLOD和LLOQ确定为0.5和1.0 log（10）/ mL，以及1.4和22 log（10）/ mL。 qRT-PCR测定的测定间重复性（4.2％变异系数[CV]）高于感染性测定（7.9％CV）。我们已经表明，这两种测定法密切相关（r = 0.85，P <0.05，n = 22）。颗粒/感染率之比确定为66。两种测定均使用色谱和过滤过程的病毒清除率样品评估病毒去除率。在这里，我们证明了qRT-PCR分析的测试速度要快得多，并且其敏感性比感染性分析高8倍。因此，qRT-PCR测定法可以在许多情况下代替传染性测定法，但是这两种测定法在阐明病毒灭活验证中的病毒灭活和去除机理方面是互补的。 （C）2015年国际生物标准化联盟。由Elsevier Ltd.出版。保留所有权利。

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