Bonvicini Francesca, Mirasoli Mara, Gallinella Giorgio, Zerbini Marialuisa, Musiani Monica, Roda Aldo
Department of Clinical and Experimental Medicine, University of Bologna, Via Massarenti 9, I-40138 Bologna, Italy.
Analyst. 2007 Jun;132(6):519-23. doi: 10.1039/b701664f. Epub 2007 Mar 30.
To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection. The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection. The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.
为了在感染后不同时间对单个感染细胞中的细小病毒B19核酸进行超灵敏定位和定量检测，已开发出一种基于肽核酸（PNA）的原位杂交（ISH）检测方法，并采用化学发光检测。该检测方法基于使用生物素标记的PNA探针，通过链霉亲和素连接的碱性磷酸酶和化学发光二氧杂环丁烷磷酸衍生物底物进行检测。发光信号通过连接到落射荧光显微镜的超灵敏液氮冷却电荷耦合器件（CCD）相机进行定量和成像。该检测方法用于分析细胞培养物中的细小病毒B19感染过程，并对感染后不同时间的病毒核酸量进行定量。化学发光ISH-PNA检测方法的特点是分辨率高，能在单个细胞内对B19核酸进行精确定位，相对于传统的比色ISH检测具有更高的灵敏度。由于化学发光检测具有高检测能力和宽线性范围，能够准确评估感染细胞百分比，其在感染后24小时达到最大值，遵循B19病毒感染周期。化学发光检测还允许在单细胞水平对病毒核酸进行定量分析，显示感染细胞中病毒核酸含量在感染后随时间持续增加。因此，所开发的化学发光ISH-PNA检测方法可成为评估病毒感染以及在病毒研究和诊断中对感染细胞的病毒核酸载量进行定量评估的有力工具。
