Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti
Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti
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DOI：
10.4269/ajtmh.2006.74.132
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发表时间：
2006-01-01
影响因子：
3.3
通讯作者：
Black, W
中科院分区：
文献类型：
--
作者：
Richardson, J;Molina-Cruz, A;Black, W
在来自墨西哥切图马尔的登革病毒-2敏感株感染后14天的血餐(Dpi)中，从埃及伊蚊个体的中肠和腿中定量检测到登革病毒-2(DENV-2)RNA。建立了一种基于SYBR Green I的实时定量逆转录聚合酶链式反应(RT-PCR)方法。检测下限和定量下限分别为每次反应20和200个拷贝。正链和负链病毒RNA链的数量是相关的。在C6/36细胞培养物和蚊子中，空斑形成单位数(PFU)与DENV-2 RNA拷贝数相关。PFU始终比RNA拷贝数低2-3log(10)。中肠DENV-2 RNA水平最高为8dpi，并在6-9dpi之间不规则波动。在感染的蚊子中，DENV-2 RNA的拷贝在每个时间点都有显著差异。定量实时RT-PCR是一种方便和可靠的方法，为病毒与媒介的相互作用提供了新的见解。
Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.