In Situ Detection of Adenovirus DNAand mRNA in Individual Cells

摘要

Infection by DNA viruses such as human adenoviruses (HAdVs) causes a highlevelaccumulation of viral DNA and mRNA in the cell population. However,the average viral DNA and mRNA content in a heterogeneous cell populationdoes not inevitably reflect the abundance in individual cells. As the vast majorityof virus infection studies is carried out using standard experimental procedureswith heterogeneous cell populations, there is a need for a method allowingsimultaneous detection and quantitative analysis of viral genome accumulationand gene expression in individual infected cells within a population. Thisarticle describes a padlock probe–based rolling-circle amplification protocolthat allows simultaneous detection of HAdV type 5 (HAdV-5) DNA and variousvirus-encoded mRNAs, as well as quantitative analysis of HAdV-5 DNA copiesand mRNA species, in individual cells within a heterogeneous population. Thisversatile method can be used to detect the extent of pathogenic DNA virusinfection in different cell types over prolonged infection times. Furthermore,simultaneous viral DNA and mRNA quantification in individual cells allowsidentification of cells in which persistent infections may be established. C 2018by John Wiley & Sons, Inc.