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Double-layer plaque assay for quantification of enteroviruses
Laura Mocé-Llivina1, Francisco Lucena, Juan Jofre
1Department of Microbiology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Applied and Environmental Microbiology
|May 7, 2004
Summary
A novel double-layer plaque assay significantly enhances enterovirus quantification. This improved method yields approximately tenfold higher virus counts compared to traditional monolayer plaque assays, aiding in better detection of naturally occurring viruses.
Area of Science:
- Virology
- Microbiology
- Environmental Science
Background:
- Accurate quantification of enteroviruses is crucial for public health and environmental monitoring.
- Existing virus quantification methods, such as the monolayer plaque assay, often underestimate viral load.
Purpose of the Study:
- To introduce and evaluate a novel double-layer plaque assay for improved enterovirus quantification.
- To compare the efficiency of the double-layer plaque assay against other established virus quantification methods.
Main Methods:
- A double-layer plaque assay was developed by combining monolayer and suspended-cell plaque assay techniques.
- The assay was validated using both pure virus cultures and naturally occurring enteroviruses in water samples.
- Cell pretreatment with 5-iodo-2'-deoxyuridine (IDU) and the use of specific cell lines (CaCo2) were investigated.
Main Results:
- The double-layer plaque assay consistently yielded significantly higher enterovirus counts, approximately one order of magnitude greater than the monolayer plaque assay.
- The ranking of assay efficiency was determined as: double-layer plaque assay >= suspended-cell plaque assay > membrane filter counting >= most probable number > monolayer plaque assay.
- Using the CaCo2 cell line and IDU pretreatment further enhanced the recovery of naturally occurring enteroviruses.
Conclusions:
- The double-layer plaque assay represents a substantial advancement in enterovirus quantification, offering superior sensitivity and recovery rates.
- This method facilitates the detection of a broader range and higher numbers of enteroviruses, particularly in environmental samples.
- The double-layer plaque assay, especially with IDU pretreatment and specific cell lines, is recommended for more effective virus detection and monitoring.