Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture.
A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.
Qin
Qi Wei
QW
State Key Laboratory for Biocontrol, College of Life Science, Zhongshan University, 135 West Xingang Road, Guangzhou 510275, PR China. tmsqingw@nus.edu.sg
Gin
Karina Yew-Hoong
KY
Lee
Li Yen
LY
Gedaria
Alice Ilaya
AI
Zhang
Sheng
S
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D017994:Iridovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D010473:Perciformes / Q000821:virology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral