A fast on-site quarantine for the detection of amphibian infection of ranavirus

초록

Background: The ranavirus outbreak has caused high mortality in mostly amphibian species, which has led to the amphibian extinction and ecological imbalance. Besides the conventional method for ranavirus detection, some isothermal methods, including loop-mediated isothermal amplification and recombinase polymerase amplification, have also been introduced. However, previous techniques face drawbacks such as lengthy analysis and indistinguishable viral names due to similar gene sequences. Therefore, fast on-site identification of ranavirus is crucial to prevent its deadly spread, and polymerase chain reaction (PCR) is typically used to detect infected species. Result: This study developed a method to precisely discriminate two ranavirus strains-frog virus 3 (FV3) and tiger frog virus (TFV)-for fast on-site quarantine via single nucleotide polymorphism (SNP) discrimination using allele-specific PCR (AS-PCR). Due to high gene sequence similarity, detection was achieved by designing a common reverse primer for FV3 and TFV, making three primers sufficient for multiplex detection. The reaction took less than 40 min and showed high specificity and sensitivity, identifying FV3 at 101 bp and TFV at 223 bp with a detection limit of approximately 1 ng/mu L of genetic material. Moreover, a visible readout for amphibians infected with FV3, TFV, or both was confirmed by a color change in silver nanoparticle formation under UV illumination from a DNA-promoting silver nitrate solution. Significance: The research illustrates a short analysis for the specific detection of ranavirus strains among frog infections in one pot, which has rarely been described before. Hence, this approach enables rapid on-site quarantine of infected amphibians, with broad applications in amphibian trade and conservation at border gates, wildlife sanctuaries, and farms.

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