Visual and rapid detection of Plesiomonas shigelloides using loop-mediated isothermal amplification method.

Area of Science:

• Aquatic microbiology
• Molecular diagnostics
• Veterinary medicine

Background:

• Plesiomonas shigelloides is a significant pathogen in aquaculture, causing disease in aquatic animals.
• Accurate and rapid diagnostic methods are crucial for managing P. shigelloides infections in fish populations.

Purpose of the Study:

• To develop a loop-mediated isothermal amplification (LAMP) method for the visual detection of P. shigelloides.
• To evaluate the sensitivity and accuracy of the LAMP method compared to conventional PCR for diagnosing P. shigelloides in aquatic animals.

Main Methods:

• Loop-mediated isothermal amplification (LAMP) was optimized at 64°C for 50 minutes to detect P. shigelloides DNA.
• Amplified products were visualized using calcein or nucleic acid dipstick assay (NADA), eliminating the need for gel electrophoresis.
• The LAMP assay's limit of detection was determined to be 2.0 × 10^2 copies/μl for recombinant plasmids.

Main Results:

• The LAMP method demonstrated a higher detection rate (42.4%) than conventional PCR (34.1%) in 85 fish specimens.
• The limit of detection for LAMP was ten times higher than conventional PCR.
• Visual detection of amplified products was achieved without agarose gel electrophoresis.

Conclusions:

• The combined LAMP, calcein, and NADA method provides a rapid, specific, sensitive, and accurate approach for detecting P. shigelloides in fish.
• This LAMP-based assay is a reliable and convenient tool for laboratory diagnosis of P. shigelloides infections in aquatic animals.
• The method offers significant advantages for aquaculture disease management and surveillance worldwide.