Development of a real-time quantitative reverse transcriptase PCR assay for detection of the Friend leukemia virus load in murine plasma
Development of a real-time quantitative reverse transcriptase PCR assay for detection of the Friend leukemia virus load in murine plasma
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DOI：
10.1016/j.jviromet.2007.10.009
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发表时间：
2008-02-01
影响因子：
3.1
通讯作者：
Li, Guo-Qiao
中科院分区：
文献类型：
--
作者：
He, Jin-Yang;Cheng, Hui-Jun;Li, Guo-Qiao
Friend白血病病毒（FLV）是一种小鼠逆转录病毒，几十年来一直被用作阐明人类免疫缺陷病毒（HIV）免疫发病机制和评价抗HIV药物效果的模型。然而，目前还没有直接检测血浆病毒载量的方法。本研究建立了TaqMan实时定量逆转录酶PCR （qRT-PCR）方法，用于快速检测和定量FLV。通过从FLV包膜基因内合成标准RNA以生成标准曲线来测量绝对FLV载量。该分析允许定量范围从20到2 × 10(8) RNA拷贝每个反应在两步实时定量逆转录酶PCR协议。探讨初始注射FLV剂量与血浆FLV载量及脾脏指数的关系。随后，评估齐多夫定、阿德福韦酯和恩替卡韦对感染FLV的小鼠的体内作用。结果表明，血浆FLV载量与脾脏指数在相同FLV注射剂量系列中不成正比，尽管观察到一种趋势。当使用血浆病毒载量进行评估时，高剂量（15mg /(kg d)）阿德福韦酯能够显著抑制小鼠FLV复制。本文所描述的qRT-PCR检测可以特异、灵敏和直接地检测FLV，也可以提供更精确的FLV负载测量。(c) 2007 Elsevier B.V.版权所有
Friend leukemia virus (FLV), a murine retrovirus, has been used as a model for elucidation of human immunodeficiency virus (HIV) immunopathogenesis and evaluation of anti-HIV drug effects for several decades. However, no method for direct detection of the plasma viral load has yet been reported. In this study, a TaqMan real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was established for the rapid detection and quantitation of FLV. Measurement of the absolute FLV load was achieved through synthesis of a standard RNA from within the FLV envelope gene for generation of a standard curve. The assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction in a two-step real-time quantitative reverse transcriptase PCR protocol. The relationships between the initially injected FLV dose and the plasma FLV load and spleen index were explored. Following this, the in vivo effects of zidovudine, adefovir dipivoxil, and entecavir on mice infected with FLV were evaluated. The results showed that the plasma FLV load was not proportional to the spleen index over the same FLV injection dosage series, although a trend was observed. When evaluated using plasma viral load, high dose (15 mg/(kg d)) adefovir dipivoxil was capable of significant inhibition of FLV replication in mice. The qRT-PCR assay described here allows specific, sensitive and direct detection of FLV and may also provide more precise measurement of FLV load. (c) 2007 Elsevier B.V. All rights reserved.