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Updated: Jun 24, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Development and Validation of a Multienzyme Isothermal Rapid Amplification-Based Fluorescence Assay for Detection of
Eun Seo Lee1, Guk Hyun Kim1, Kwang Il Kim1
1Department of Aquatic Life Medicine, Pukyong National University, Busan, Republic of Korea.
Abstract:
Megalocytivirus pagrus 1 is a major viral pathogen that causes high mortality and economic losses in aquaculture worldwide. Although PCR-based diagnostics are highly sensitive and specific, their equipment dependence and long assay times limit field applicability. In this study, a digital multienzyme isothermal rapid amplification with exonuclease probe (digital MIRA-EXO) assay was developed and validated for rapid and specific detection of red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The digital MIRA-EXO assay specifically identified Megalocytivirus pagrus 1 without cross-reactivity with non-target samples, including four fish cell lines and 13 different fish pathogens. Analytical sensitivity, expressed as 95% limit of detection, ranged from 146.87 to 201.6 copies/μL across the three viruses. Diagnostic performance evaluation of 180 fish samples showed high sensitivity (92.22%), specificity (100%), and overall accuracy (96.11%), with an area under the receiver operating characteristic (ROC) curve of 0.971. Substantial agreement with reference assays was observed for experimentally infected (κ = 0.786) and field samples (κ = 0.792), and between column-based and rapid DNA extraction methods (κ = 0.783). Overall, the digital MIRA-EXO assay provides a rapid, accurate, and field-deployable diagnostic tool for Megalocytivirus pagrus 1 detection.
Insights
A new digital multienzyme isothermal rapid amplification with exonuclease probe (MIRA-EXO) assay offers rapid and accurate detection of Megalocytivirus pagrus 1, a significant aquaculture pathogen. This field-deployable tool addresses limitations of traditional PCR methods for fish disease diagnostics.
Area of Science:
- Aquatic Animal Health
- Molecular Diagnostics
- Virology
Background:
- Megalocytivirus pagrus 1 causes significant mortality and economic losses in global aquaculture.
- Current PCR-based diagnostics are sensitive and specific but impractical for field use due to equipment and time constraints.
Purpose of the Study:
- To develop and validate a rapid, field-deployable diagnostic assay for Megalocytivirus pagrus 1.
- To detect specific iridoviruses including red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV).
Main Methods:
- Development of a digital multienzyme isothermal rapid amplification with exonuclease probe (digital MIRA-EXO) assay.
- Validation using non-target samples (fish cell lines, fish pathogens) and fish samples.
- Performance evaluation against reference assays and different DNA extraction methods.
Main Results:
- The digital MIRA-EXO assay demonstrated high specificity, with no cross-reactivity against non-target organisms.
- Analytical sensitivity ranged from 146.87 to 201.6 copies/μL.
- Diagnostic evaluation showed high sensitivity (92.22%), specificity (100%), and accuracy (96.11%) with an ROC AUC of 0.971.
- Substantial agreement was found with reference assays and between DNA extraction methods.
Conclusions:
- The digital MIRA-EXO assay is a rapid, accurate, and specific tool for detecting Megalocytivirus pagrus 1.
- This assay is suitable for field deployment, overcoming limitations of conventional PCR.
- It offers a valuable solution for managing viral diseases in aquaculture.
