An indicator cell assay for detection of human cytomegalovirus based on enhanced green fluorescent protein.
An indicator cell line (ML-UL54-EGFP) for the detection of human cytomegalovirus (HCMV) by a simple and direct method was developed. The stable line was constructed by introducing into mink lung cells an expression cassette that contains the enhanced green fluorescent protein (EGFP) reporter gene under the control of an HCMV-inducible promoter. The promoter was from the upstream region of the HCMV UL54 (pol) gene, an early gene promoter that is activated in the early phase of HCMV infection. Following infection with HCMV for 48 h, the stable line expressed well detectable level of the EGFP as observed under a fluorescence microscope. The sensitivity of the indicator cell assay is at least comparable with that of a plaque assay as assessed with a panel of HCMV strains. There were no detectable fluorescent cells after inoculations with several viruses other than HCMV, indicating high specificity. Analysis with flow cytometry revealed that the induced fluorescence from the infected cells was proportional to the titer of HCMV inoculated, making it possible to quantify HCMV infectious particles. In summary, the EGFP-based indicator cell line is of potential use for rapid detection and quantification of HCMV in clinical specimens.
Liu
W T
WT
Faculty of Medical Technology and Institute of Biotechnology in Medicine, National Yang-Ming University, Shih-Pai, 112, Taipei, Taiwan, ROC.
Sun
J R
JR
Lin
C H
CH
Kuo
R L
RL
Kung
S H
SH
D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000254:growth & development* / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000821:virology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008907:Mink; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D014764:Viral Proteins
D008164:Luminescent Proteins; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase