Quantitative Detection Of White Spot Syndrome Virus Using Gold Nanoparticle Probe-based Dark-Field Microscopy

White spot syndrome(WSS),caused by white spot syndrome virus(WSSV),is one of the most widespread and harmful diseases,which is a serious threat to the health development of shrimp industry.At present,there are no effective drugs for the treatment of shrimp infected with the WSSV.So early detection of WSSV and timely removal of the infected shrimp and purification of aquaculture water are the most effective ways to prevent the outbreak of WSS.Besides,the diffusion rate of WSSV is very fast,so a simple and quick detection technology is urgently needed.Gold nanoparticles have many advantages,such as excellent plasma resonance effect,good biocompatibility and highly modifiable surface.With dark-field microscope imaging technology,gold nanoprobes modified with antibodies can be used to label the target pathogens and make them visible under a dark-field microscope due to the excellent plasma resonance property of gold nanoparticle.In this study,we provide a gold nanoprobes assisted dark-field microscope count for rapid and quantitative detection of the WSSV in samples.This work consists of the following three parts:(1)Proliferation,purification and quantification of WSSV:WSSV was isolated and purified by sucrose density gradient centrifugation from the homogenated tissues of WSSV-infected procambarus clarkii(crayfish).The collected WSSV was assayed with transmission electron microscopy(TEM).The results showed that the purification is successful,and the envelope of most WSSV is broken,leaving the nucleocapsid exposed.The conserved sequence of nucleocapsid gene VP664 of WSSV was obtained by PCR with the specific primers for the genome of the virus and was inserted into the plasmid pET-28a as standard materials.Fluorescence quantitative PCR was used to construct the standard relationship curve of between concentrations and Ct values.The relationship equation between Ct value and plasmid copy number(X)is Ct=-3.7168X+42.291(X was the logarithm of plasmid copy number),and the correlation coefficient R2=0.9991,indicating that the standard curve has a good linear relationship.The concentration of the purified WSSV stock is determined to be 7.12×107 copies/μL by the quantitative PCR.(2)Preparation and identification of anti-WSSV VP664 polyclonal antibody:In this study,a highly conserved sequence of the WSSV VP664 gene was cloned into the pET-28a(+)expression vector,and the WSSV VP664 protein was successfully expressed and purified using E.coli BL21 as the host bacteria.The recombinant protein was then used as immunogen to immunize New Zealand white rabbits.The serum titer was determined to be 1:256000 by ELISA.After purification of the serum,the prepared polyclonal antibody was detected to be specific to WSSV by Western blot.(3)Construction of gold nanoprobe combined with dark field microscope for detection of WSSV:1)Preparation and characterization of gold nanoprobe:The prepared polyclonal antibodies were co-incubated with gold nanoparticles(with a diameter of 15 nm).The purified gold nanoprobes were obtained by several centrifugation washings.The resultant probes were analyzed by SDS-PAGE,UV-Vis absorption spectrum,DLS and TEM.The results showed that the gold nanoparticles were successfully modified with antibodies and has a good monodispersion.There are about 50 antibody molecules on the molecules binding the surface of each gold nanoparticle.2)Gold nanoprobes assisted dark-field microscopy detection of WSSV:Purified WSSV was used to verify our gold nanoprobes assisted dark-field microscopy counting method.After gold nanoprobes were incubated with the purified WSSV,the bright golden elliptical structure could be clearly observed under the dark-field microscope,while the individual gold nanoprobe and WSSV without gold nanoprobes are not visible under dark-field.TEM observation showed that the WSSV was surrounded by hundreds of gold nanoprobes.Furthermore,the LOD(limit of detection)of our counting method was investigated with the serial diluted WSSV samples.The results show that the detection limit was about 5.7×101 copies/μL,and a sensitivity 10 times higher than that of fluorescence quantitative PCR.Finally,we also detected the real samples of crayfish infected with WSSV,and the results showed that the quantitative detection results of WSSV from real samples by gold nanoprobes assisted dark-field microscopy are consistent with the date of fluorescence quantitative PCR.Therefore,the gold nanoprobes assisted dark-field microscope counting method for detection of WSSV have advantages such as simple in preparation of the probe,rapid detection and the sensitive.In conclusion,the gold nanoprobes assisted dark-field microscopy counting established in this paper has the advantages of simple operation,fast detection(sample prepared via one-step incubation(mixing the supernatant of shrimp tissue with the probe for 30 minutes)was subjected to imaging under dark-field microscope,and subsequently counting with software automatically)and high sensitivity(10 times more sensitive than qPCR).This work provides a new technique for the ultra-sensitive and rapid detection of WSSV,serving as an effective strategy for the prevention of WSS.