Civil and Environmental Engineering, University of California, Irvine, California 92697, USA.
Appl Environ Microbiol. 2010 Mar;76(5):1442-8. doi: 10.1128/AEM.01937-09. Epub 2010 Jan 15.
Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10(4) PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r(2) value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r(2) value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.
方法快速检测和定量环境中的传染性病毒是迫切需要的公共卫生保护。荧光激活细胞分选（FACS）分析被开发出来，以检测传染性腺病毒（Ads）基于病毒蛋白的表达复制过程中的细胞。该试验是首次采用重组腺病毒血清型 5 （rAd5）与 E1A 基因的绿色荧光蛋白（GFP）基因取代。细胞感染 rAd5 表达 GFP，这是捕获和通过 FACS 定量。结果表明，rAd5 可以检测到浓度为 1 到 10（4）PFU 每个试验在 3 天内，显示出之间的线性关系病毒浓度和 GFP 阳性细胞数与 r（2）值大于 0.9。按照同样的概念，荧光标记的抗体分别针对 E1A 和六邻体蛋白的 FACS 检测法被开发出来。针对六邻体的检测法比针对 E1A 的检测法更灵敏。结果表明，在 3 天内，基于六邻体蛋白，FACS 可以检测到低至 1 PFU 的 Ads，r（2）值在 4 个对数浓度范围内大于 0.9。将该方法应用于环境样品表明，在 50%的原污水样品和 33%的二级处理污水样品中检测到传染性 Ads 呈阳性，但在 12 个海水样品中均未发现。传染性 Ads 的数量在 10 到 165 PFU/100 毫升污水样品之间。结果表明，FACS 分析是一种快速定量检测环境样品中传染性 Ads 的工具，也代表了传染性病毒快速环境监测的重大进展。
