Eisler Diane L, McNabb Alan, Jorgensen Danielle R, Isaac-Renton Judith L
Division of Laboratory Services, British Columbia Centre for Disease Control, Vancouver, British Columbia, Canada.
J Clin Microbiol. 2004 Feb;42(2):841-3. doi: 10.1128/JCM.42.2.841-843.2004.
We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively.
我们报告了使用西尼罗河病毒装甲RNA作为内部阳性对照（IPC），用于从野外采集的蚊子中提取RNA以及进行逆转录聚合酶链反应（RT-PCR），并报告了一种多重实时Taqman RT-PCR方法，用于同时检测西尼罗河病毒的3'非编码区和包含IPC的西尼罗河病毒NS5-2区。对来自加拿大不列颠哥伦比亚省的蚊子样本池（n = 635）进行了重复检测，结果显示西尼罗河病毒呈阴性，IPC呈阳性。对来自艾伯塔省和曼尼托巴省的已知西尼罗河病毒阳性蚊子样本池的上清液进行了重复检测，结果显示西尼罗河病毒基因组的两个区域均呈阳性。在批量提取对照中，IPC的平均循环阈值（Ct）值±2个标准差为36.43±1.78个循环。98.4%（624）的西尼罗河病毒阴性样本池的IPC值落在该范围内，表明对于不同蚊子属和数量的样本池，RNA提取和RT-PCR具有可重复性。对蚊子样本池属的比较显示，对IPC的Ct值没有显著的属效应。将西尼罗河病毒装甲RNA用作IPC可分别监测RNA提取和RT-PCR，并检测由于这些过程失败或PCR抑制导致的假阴性结果。
