Study On The Detection Technology Of Immunocolloidal Gold Test Strips Of Crab Snails

Spiroplasma eriocheiris is a new kind of emerging pathogen in commercially exploited freshwater crustaceans.Besides Chinese mitten crab Eriocheir sinensis,other freshwater crustaceans,the white shrimp Penaeus vannamei,prawns Macrobrachium rosenbergii and Macrobrachium nipponense,as well as crayfish Procambarus clarkiaμl were also the hosts of spiorplasma,indicating that spiroplasmas have become serious,novel pathogens that can potentially threaten global aquacμlture of crustaceans.Therefore,spiroplasma disease control has become a worldwide priority for achieving sustainable crustacean production,in which diagnosis and epidemiological surveys play a key role.Some methods,such as clinical signs,light microscopy,electron microscopic histopathology,in situ hybridization,PCR-based molecμlar diagnostics and real-time PCR with SYBR Green chemistry have been effectively used for detection of the spiroplasmas from aquatic crustaceans.However,these methods are not suitable for on-site fast detection in aquacμlture farms because of the requirement of highly qualified personnel,special equipment and expensive reagents.Althoμgμgh Enzyme-liked immunosorbent assay can be used for detection of the spiroplasmas in aquacμlture farms,it takes about 150 min and requires a long incubation time and tedious washing steps.In the present study,we represented the study of the immunochromatographic strip.The resμlts of an immunochromatographic strip can be examined visually,thus providing fast and simple on-site detection without the need of skilled personnel and any instruments.Main results of this dissertation included the following two parts.1.Production of polyclonal antibody against Spiroplasma eriocheirisIn this paper,on the basis of preliminary studies,through the use of traditionalimmunization methods we made four times immunization,and took blood a week later.We also explored best preparation methods of high titer polyclonal antibody against pathogens.The traditional methods of indirect ELISA was used to test the titer,4 times the gradient dilution to 1:97152,and dilution ratio of Goat Anti-rabbit IgG is at 1:3000.However,by affinity determination method and using sigmaplot software for data simulation,affinity constants are obtained.The result showed that the titer of antibody against the pathogen was 1:31250;The affinity constant is 4× 108.2.Preparation of immunochromatographic test stripUse trisodium citric acid deoxidization to prepare colloidal gold,and then it was coupled with rabbit polyclonal antibody of Spiroplasma eriocheiris.Using double antibody sandwich colloidal gold immune chromatography,The polyclonal antibody of Spiroplasma eriocheiris is screw at test line and Goat Anti-rabbit IgG is at control line.The immunochromatographic test strip is preliminarily established to detect Spiroplasma eriocheiris.The results showed that the preparation of colloidal gold is 40 nm,maximum absorption peak is 522 nm,the optimal pH is 8 and concentration is 15 ug/ml.0.01M pH7.2 phosphate buffer solution(PBS)is used to be dilution.The results of immunochromatographic strip can be examined visually,thus providing fast and simple on-site detection and the limit of detection was found to be 104CCU-ml-1.