Development And Application Of A Quantum Dot Microsphere Immunochromatographic Assay For Detection Of Africa Swine Fever Virus Antibodies

African swine fever(ASF)is a highly contagious animal disease caused by African swine fever virus(ASFV),which can infect domestic pigs and wild boars at the same time.Acute ASF has the characteristics of rapid onset,short course of disease,and high mortality.The specific manifestations are high fever,loss of appetite,and severe internal organ bleeding.Since August 2018,China’s first case of ASF broke out in Shenyang,and within a few months ASF quickly swept most of China’s provinces,posing a huge threat to China’s aquaculture industry and causing serious economic losses.At present,there is no commercial ASFV vaccine and effective treatment,the spread and outbreak of the disease are mainly controlled by strict disinfection measures and slaughtering quarantine.It is very important to detect ASFV quickly and sensitively for disease prevention and control.The main methods of ASFV diagnosis are virus isolation,Polymerase chain reaction(PCR),Real-time quantitative polymerase chain reaction(real-time quantitative PCR)and fluorescent antibody test.However,these methods all require expensive instruments and professional operators,and take a long time,so they are not suitable for on-site testing.In areas where the ASF outbreak occurs,surviving animals(especially those affected by subacute and chronic infections)may maintain detectable antibody levels after infection and can serve as virus hosts.It is an important indication to detect the antibody of the surviving ASFV host in the epidemic area to prove that the virus can still spread in the area.Therefore,monitoring ASFV specific antibodies is of great significance for the host infection status and the evaluation of vaccine immune effect in the future.The traditional Enzyme linked immunosorbent assay(ELISA)can detect ASFV-specific antibodies in infected pigs.However,the ELISA involves many steps,time-consuming and laborious,and the operators need to receive certain professional training.Immunochromatographic assay(ICA)is widely used in point-of-care testing(POCT)due to its simple operation,with no need for professionals and precision instruments.As a traditional marking material,colloidal gold is often used in ICA,but the sensitivity of colloidal gold ICA is low.Therefore,there is an urgent need for a simple,efficient and rapid method to detecte ASFV antibodies.In this study,quantum dot fluorescent microsphere was used as a marker,indirect method was combined with immunochromatographic technique,and ASFV P30 protein with high sensitivity and early expression was selected as the labeled protein,and quantum dot fluorescent microsphere immunochromatographic test strip was established.The fluorescence intensity of the test line(T line)and the control line(C line)of the immunochromatographic strip were read by the fluorescence immunoanalyzer instrument,and calculate the ratio of T line to total(T line and C line)fluorescence value(FI_T/(FI_T+FI_C)),which effectively avoiding the error caused by subjective observation by the naked eye.After optimizing the relevant parameters of the experimental system,the on-site rapid detection was achieved within 15 minutes.The detection limit of this experimental detection system for ASFV antibody is 1:32000dilution,and there is a good linear relationship in the 1:1000-1:16000 dilution interval,R~2 is0.99452.There was no cross reaction in the positive sera of porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus-2(PCV2),pseudorabies virus(PRV),foot-and-mouth disease virus(FMDV),classical swine fever virus(CSFV),haemophilus parasuis-2(HPS2),haemophilus parasuis-8(HPS8),haemophilus parasuis-10(HPS10),mycoplasma hyopneumoniae(Mhp),mycoplasma hyosynoviae(Mhs),mycoplasma flocculara(Mf)and swine influenza virus H3N2(H3N2)detected on the immunochromatographic strip,which indicated that the method had high specificity.The repeatability verification of the same batch and different batches of immunochromatographic test strips showed that the coefficient of variation was less than 10%,which proved that the method has good repeatability.The shelf life test results show that the immunochromatographic test strips prepared by this method can still maintain the corresponding detection performance after being stored at 4℃,25℃and 37℃for 3 months.Compared with commercial ELISA kit and colloidal gold strip,the sensitivity of this method is superior.Finally,the quantum fluorescent microsphere immunochromatographic test strip was used to detect 322 clinical serum samples,with an accuracy rate of 98.45%,indicating that this method has practical clinical application value.