Establishment and application of in situ LAMP for detection of Haliotid herpesvirus 1 (HaHV-1)

To elucidate the tissue tropism and spreading routes of Haliotid herpesvirus 1 (HaHV-1) in susceptible Haliotis diversicolor aquatilis after infection, an in situ loop-mediated isothermal amplification (LAMP) method for HaHV-1 was developed in this study based on LAMP and in situ hybridization techniques. The developed in situ LAMP method was then applied on pedal nerve branches, mantle, hepatopancreas and gill of specimens collected across the experimental infection process. For the establishment of the HaHV-1 in situ LAMP detection method, we firstly optimized LAMP primers for specific detection of HaHV-1, improving the specificity and stability of the LAMP reaction on slides. We then optimized the time for color development (about 60 min) on pedal nerve branches, which was identified as the most suitable target for pathological investigation and in situ LAMP detection. Finally, the HaHV-1 in situ LAMP method was established after color development with immunoenzymatic labeling technique. The results showed that the incorporation of the designed loop primers yielded stabile results during LAMP amplification. The developed in situ LAMP detection method was applied on major tissue collected at 24、36、48、60 and 72 h across the experimental infection process. The in situ LAMP signals were firstly detected at 36 h on pedal nerve branches, and at 48 h on mantles after infection. The viral signals detected at mantle were confined to the peripheral nerves scattered throughout the mantle. Finally, in situ LAMP signals were identified occasionally in connective tissue of hepatopancreas of samples collected at the end of the experimental infection. The viral infection signals were always accompanied by tissue lesions and infiltrated haemocytes infected with HaHV-1. The in situ LAMP method developed in this study achieved the goal of rapid detection and definite diagnosis of HaHV-1, and is helpful for investigating the tissue tropism and pathological characteristics in different susceptible species. The detection method constitutes a potentially valuable tool for the diagnosis of HaHV-1 infection, and studies associated with tissue tropism, invasion pathways and pathogenic mechanism of HaHV-1.