Kothera Linda, Byrd Brian, Savage Harry M
Centers for Disease Control and Prevention, 3156 Rampart Rd., Fort Collins, CO 80521.
Western Carolina University, CHHS 416, 3971 Little Savannah Road, Cullowhee, NC 28723.
J Med Entomol. 2017 Nov 7;54(6):1567-1572. doi: 10.1093/jme/tjx125.
Aedes aegypti (L.) and Ae. albopictus (Skuse) are important arbovirus vectors in the United States, and the recent emergence of Zika virus disease as a public health concern in the Americas has reinforced a need for tools to rapidly distinguish between these species in collections made by vector control agencies. We developed a duplex real-time PCR assay that detects both species and does not cross-amplify in any of the other seven Aedes species tested. The lower limit of detection for our assay is equivalent to ∼0.03 of a first-instar larva in a 60-µl sample (0.016 ng of DNA per real-time PCR reaction). The assay was sensitive and specific in mixtures of both species that reflected up to a 2,000-fold difference in DNA concentration. In addition, we developed a simple protocol to extract DNA from sonicated first-instar larvae, and used that DNA to test the assay. Because it uses real-time PCR, the assay saves time by not requiring a separate visualization step. This assay can reduce the time needed for vector control agencies to make species identifications, and thus inform decisions about surveillance and control.
埃及伊蚊（Aedes aegypti (L.)）和白纹伊蚊（Ae. albopictus (Skuse)）是美国重要的虫媒病毒传播媒介，近期寨卡病毒病在美洲成为公共卫生问题，这凸显了在病媒控制机构采集的样本中快速区分这两种蚊子的工具的必要性。我们开发了一种双重实时聚合酶链反应检测法，该方法能同时检测这两种蚊子，且在测试的其他七种伊蚊中均无交叉扩增现象。我们检测法的检测下限相当于在60微升样本中约0.03条一龄幼虫（每个实时聚合酶链反应检测的DNA为0.016纳克）。该检测法在两种蚊子的混合样本中灵敏且特异，样本中DNA浓度差异高达2000倍时仍能检测。此外，我们开发了一种从经超声处理的一龄幼虫中提取DNA的简单方法，并使用该DNA测试检测法。由于采用实时聚合酶链反应，该检测法无需单独的可视化步骤，节省了时间。这种检测法可以减少病媒控制机构进行物种鉴定所需的时间，从而为监测和控制决策提供依据。
