Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors.
Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors.
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DOI：
10.1371/journal.pone.0050859
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Verhasselt B
中科院分区：
文献类型：
--
作者：
Vermeire J;Naessens E;Vanderstraeten H;Landi A;Iannucci V;Van Nuffel A;Taghon T;Pizzato M;Verhasselt B
在不同的实验室和应用中，需要对细胞培养上清液和其他生物制剂中的逆转录病毒进行定量。基于抗原检测（例如HIV的p24）或基于基因组检测的方法是病毒特异性的，并且有时受到检测的有限动态范围的影响。相反，逆转录酶（RT）活性的测量是一种通用方法，可采用实时PCR定量（基于qPCR的产物增强RT（PERT）测定）进行调整以获得更高的灵敏度。我们使用市售试剂（如MS 2 RNA和即用型qPCR混合物）对改良的SYBR绿色I PERT检测（SG-PERT）进行了评价。该检测试剂盒的动态范围为7 log，灵敏度为10 nU HIV-1 RT，并且通过更低的运行间变异、更低的成本和更高的线性范围而优于用于HIV滴度测定的p24 ELISA。SG-PERT值分别与基于HIV的病毒载体和具有复制能力的HIV-1制剂中的转导单位和感染单位相关。该测定还可以定量莫洛尼鼠白血病病毒衍生的载体，并且可以在不同的仪器上进行，例如Roche Lightcycler® 480和Applied Biosystems ABI 7300。我们认为该检测是一种准确、快速且相对便宜的逆转录病毒定量方法，易于在常规和研究实验室中实施。
Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.
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影响因子：
3.5
作者：
Geraerts M;Willems S;Baekelandt V;Debyser Z;Gijsbers R
通讯作者：
Gijsbers R
影响因子：
2.9
作者：
Brorson, K;Swann, PG;Stein, KE
通讯作者：
Stein, KE
影响因子：
6.4
作者：
Lerma, JGG;Yamamoto, S;Heneine, W
通讯作者：
Heneine, W
影响因子：
8.2
作者：
Clémenceau, B;Jégou, D;Saï, P
通讯作者：
Saï, P
DOI：
10.1073/pnas.0306764101
发表时间：
2004-03-23
影响因子：
11.1
作者：
Levy, DN;Aldrovandi, GM;Shaw, GM
通讯作者：
Shaw, GM