Dark-field visual counting of white spot syndrome virus using gold nanoparticle probe

White spot syndrome virus (WSSV) is an important pathogen in shrimp farming and causes serious economic losses in the shrimp industry. Early detection of WSSV is particularly important in the prevention and treatment of the disease. However, the current routine technology lacks the convenience, ultra-sensitivity and speed that have limited the development of on-site detection application of WSSV. Based on the light scattering charac-teristics of gold nanoparticles (GNP) under a dark field microscope, we develop here a GNP-labelled dark-field counting method to count WSSV by the naked eye. The principle is as follows: WSSV is recognized by an anti-WSSV polyclonal antibody functionalized GNP probes (GNP probes). Therefore, hundreds of GNP probes will bind to the surface of each WSSV, forming a golden-elliptical structure visible to the naked eye under a dark field microscope because GNP has a strong plasmon resonance effect. For the detection of WSSV in pure and real tissue samples, we found that the detection sensitivity of the GNP-labelled dark-field counting method is 32 copies/mu L, which is the same order of magnitude as that of quantitative PCR (qPCR) in this work. In addition, the detection time by GNP-labelled dark-field counting method is less than 30 min, only approximately a quarter of that by qPCR. Therefore, our GNP-labelled dark-field counting method can meet the requirements of rapid and ultra -sensitive detection of WSSV clinic samples.

Automated summary

We developed a GNP-labeled dark-field counting method for the detection of white spot syndrome virus (WSSV) in shrimp. The method utilizes the light scattering characteristics of gold nanoparticles under a dark-field microscope, allowing for visual counting of WSSV by the naked eye. The sensitivity of the method was found to be 32 copies/μL in pure and real tissue samples, and the detection time was less than 30 minutes, meeting the requirements for rapid and ultra-sensitive detection of WSSV clinic samples.