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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
A simplified PCR methodology for semiquantitatively analyzing dengue viruses
Supranee Upanan1, Arturo Cabrera-Hernandez, Maneerat Ekkapongpisit
1Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Thailand.
Japanese Journal of Infectious Diseases
|December 26, 2006
Summary
Researchers developed a rapid, simplified polymerase chain reaction (PCR) method to quantify dengue viruses. This new technique uses a DNA template mimic, offering a faster alternative to traditional plaque assays for dengue virus detection.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Traditional plaque assays for dengue virus titration are slow, labor-intensive, and costly.
- Existing alternative methods often require specialized equipment inaccessible in resource-limited settings.
Purpose of the Study:
- To develop a rapid, simplified, and cost-effective semiquantitative PCR method for dengue virus quantification.
- To create a DNA-based template mimic for PCR, eliminating the need for RNA transcripts.
Main Methods:
- A DNA fragment from dengue serotype 4 was engineered into a plasmid mimic with a deletion.
- Splicing overlap extension PCR was used to create the modified plasmid.
- The plasmid mimic was tested for competitive binding against dengue virus cDNA.
Main Results:
- The engineered plasmid mimic demonstrated competitive binding with dengue virus cDNA.
- The PCR primers were specific to dengue viruses, showing no reaction with Japanese encephalitis virus cDNA.
- Accurate quantification of dengue viruses (10^3–10^6 pfu) was achieved within a single day.
Conclusions:
- The developed semiquantitative PCR method provides a rapid and accessible alternative for dengue virus titration.
- This technique overcomes limitations of conventional plaque assays, particularly for researchers in developing nations.
- The DNA template mimic approach simplifies the process and reduces turnaround time for dengue virus quantification.
