Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8747097 | Journal of Virological Methods | 2018 | 24 Pages |
Abstract
Ostreid herpesvirus-1 (OsHV-1) is a well-known pathogen associated with high mortality rates in hatchery-reared larvae and juveniles of different bivalve species worldwide. Early, rapid and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Recombinase polymerase amplification (RPA) is a novel isothermal amplification method, which can amplify detectable amount of DNA at 37â¯Â°C-39â¯Â°C within 20â¯min. In the present study, two sets of specific primers and probes were designed for the real-time quantitative RPA (qRPA) detection of OsHV-1 DNA. The sensitivity and specificity of detection were evaluated by comparison with quantitative polymerase chain reaction (qPCR). The detection limit for qRPA assays was shown to be 5 copies DNA/reaction for the primer set ORF95, which was lower than the 100 copies required for the qPCR test. The optimal reaction temperature and time were 37â¯Â°C for 20â¯min, making this approach faster than qPCR. This is the first study to apply qPCR and qRPA methods to detect OsHV-1 in Scapharca subcrenata. The percentage of viral load sample detected by the two methods was 22% and the correlation of the two virus quantitative results was 0.8. Therefore, qRPA assays is sensitive, fast, and high-temperature independent relative to qPCR and is suitable for critical clinical diagnostics use and rapid field analysis in resource-limited settings.
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Authors
Fang Gao, Jing-Zhe Jiang, Jiang-Yong Wang, Hong-Ying Wei,