Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome...

Area of Science:

• Aquaculture pathology
• Molecular diagnostics
• Viral disease detection

Background:

• White spot syndrome virus (WSSV) is a major pathogen in crustacean aquaculture, causing significant economic losses due to mass mortality.
• Accurate and rapid detection methods are crucial for managing WSSV outbreaks and implementing effective control strategies in shrimp farming.

Purpose of the Study:

• To develop and validate a novel one-step, single-tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay for the quantitative detection of WSSV.
• To establish a precise, accurate, and sensitive diagnostic tool for WSSV detection in aquaculture settings.

Main Methods:

• Designed a set of six primers targeting eight distinct sequences of WSSV for the real-time LAMP reaction.
• Performed the assay under isothermal conditions at 63°C, with results monitored in real-time using an inexpensive turbidimeter.
• Quantification was achieved by plotting viral titre against threshold time (T(t)) using plasmid standards, establishing a standard curve (R² = 0.988).

Main Results:

• The real-time LAMP assay demonstrated high sensitivity, capable of detecting as few as 100 copies of WSSV template DNA.
• Specificity analysis confirmed no cross-reactivity with other relevant shrimp viruses (IHHNV, TSV, YHV) or healthy shrimp DNA/cDNA.
• The assay provides precise and accurate quantitative detection of WSSV.

Conclusions:

• The developed WSSV real-time LAMP assay is a valuable and efficient tool for the rapid detection and quantification of WSSV.
• This method is suitable for large-scale field sample analysis and epidemiological studies in crustacean aquaculture.
• The assay offers a promising alternative for WSSV diagnostics, contributing to improved disease management and reduced economic impact.