Richardson Jason, Molina-Cruz Alvaro, Salazar Ma Isabel, Black William
Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, Colorado, USA.
Am J Trop Med Hyg. 2006 Jan;74(1):132-41.
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.
在来自墨西哥切图马尔的登革热病毒2型（DENV-2）易感品系埃及伊蚊中，于感染性血餐（dpi）后的第14天，对每只埃及伊蚊的中肠和腿部的DENV-2 RNA进行了定量分析。开发了一种基于SYBR Green I的链特异性定量实时逆转录聚合酶链反应（RT-PCR）检测方法。最低检测限和定量限分别为每个反应20个拷贝和200个拷贝。正链和负链病毒RNA的量具有相关性。在C6/36细胞培养物和蚊子中，噬斑形成单位（PFU）的数量与DENV-2 RNA拷贝数相关。PFU始终比RNA拷贝数低2至3个对数（10）。DENV-2 RNA的中肠水平在8 dpi时达到峰值，并在6至9 dpi之间不规则波动。在每个时间点，感染蚊子中DENV-2 RNA的拷贝数差异显著。定量实时RT-PCR是一种方便可靠的方法，为病毒与载体的相互作用提供了新的见解。
