District of Columbia Department of Forensic Sciences, Public Health Laboratory Division, Immunology and Virology Unit, 401 E Street SW Washington, DC 20024, USA.
District of Columbia Department of Forensic Sciences, Public Health Laboratory Division, Immunology and Virology Unit, 401 E Street SW Washington, DC 20024, USA.
J Virol Methods. 2024 May;326:114893. doi: 10.1016/j.jviromet.2024.114893. Epub 2024 Feb 13.
West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation. This study demonstrates the successful optimization of a WNV multiplex LDT (assay targets: ENV and NS1 genes) for high-throughput environmental surveillance testing of mosquito pool homogenates on the Panther Fusion System. Analytical sensitivity of the assay was 186 and 744 copies/PCR reaction for the ENV and NS1 targets, respectively. To assess the performance of this assay, a total of 80 mosquito pools were tested, including 60 previously tested pools and 20 spiked negative mosquito pools. Among the 60 previously tested specimens, the Panther Fusion WNV LDT demonstrated 100% positive and negative agreement with the CDC West Nile RT-qPCR assay. The Panther Fusion WNV LDT also detected all 20 spiked specimens. The Panther Fusion WNV LDT assay was successfully developed and optimized for high throughput testing with similar performance to the previously used CDC West Nile RT-qPCR assay.
西尼罗河病毒（WNV）是一种通过蚊虫媒介传播的虫媒病毒。WNV 于 1999 年在美国出现，此后已成为美国的地方病，导致该国国内获得性虫媒病毒病最多。对 WNV 的蚊虫监测有助于监测随时间推移和在不同地点的虫媒病毒病负担。实时荧光定量 RT-PCR 是监测 WNV 的首选方法，但这些方法劳动强度大。 Panther Fusion 系统具有开放访问功能，允许在全自动系统上运行实验室开发的测试（LDT），用于核酸提取、实时荧光定量 RT-PCR 和结果生成。本研究证明了成功优化了一种 WNV 多重 LDT（检测靶标：ENV 和 NS1 基因），可用于 Panther Fusion 系统上的蚊虫池混合液高通量环境监测测试。该测定的分析灵敏度分别为 ENV 和 NS1 靶标 186 和 744 个拷贝/PCR 反应。为了评估该测定的性能，共测试了 80 个蚊虫池，包括 60 个先前测试过的池和 20 个加标阴性蚊虫池。在 60 个先前测试的标本中，Panther Fusion WNV LDT 与 CDC 西尼罗河实时荧光定量 RT-PCR 检测法具有 100%的阳性和阴性一致性。 Panther Fusion WNV LDT 还检测到所有 20 个加标标本。Panther Fusion WNV LDT 测定法已成功开发并优化用于高通量测试，与之前使用的 CDC 西尼罗河实时荧光定量 RT-PCR 检测法具有相似的性能。
