Construction Of Nucleic Acid Immunocolloidal Gold Probe And Its Application In PRRSV Test Strip

Porcine Reproductive and Respiratory Syndrome(PRRS)is a high-risk contact infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV can cause reproductive disorders in pregnant sows and respiratory distress in piglets,and has characteristics such as high variability,immunosuppression and persistent infection.Since its first discovery in the late 1980s,PRRSV has caused economic damage to the global pig industry for more than 30 years.Since PRRSV transitioned to China in the mid-1990s,it has become one of the main problems that endanger the healthy development of China's pig industry.To control the spread of PRRSV,the on-site diagnosis of PRRSV is particularly important.And lateral flow strips are widely used because of their low price and easy operation.However,the synthesis methods of conventional colloidal gold test strip probes are mostly based on electrostatic adsorption,the synthesis process is susceptible to various factors,and the stability is poor.Therefore,this study intends to construct nucleic acid immunocolloidal gold probes,which is expectd to improve the stability of the probes.In this study,a pair of specific primers were designed according to the ORF7 gene sequence of PRRSV R98 strain(encoding PRRSV N protein)recorded in GenBank.Then,a fragment of PRRSV N protein was amplified by RT-PCR and inserted into the pET-28a(+)vector plasmid.After that,the conditions for inducing the fragment of N protein produce in prokaryotic host such as the concentration of IPTG,the time and the temperature of induction were investigated.And the results showed that the induction with IPTG at a final concentration of 0.2 mM at 37? for 6 h could produce efficiently the recombinant protein.The successful production of the target protein was confirmed by Western blot(WB)and the high-purity recombinant protein was obtained using nickel beads enrichment method.In addition,Mass spectrometry analysis confirmed that the recombinant protein is the fragment of PRRSV N protein.Mice were immunized three times with the purified recombinant protein and the antibody titer verified by ELISA is 1:64000.The WB and immunefluorescence assay(IFA)confirmed that the polyclonal antibody can specifically react with PRRSV.Second,the DNA modified with antibody was prepared by chemical coupling,and the DNA functionalized colloidal gold nanoparticles were prepared by salt aging and freeze-thaw methods.And the nucleic acid immunocolloidal gold probe was constructed by mixing the DNA coupled antibodies with DNA functionalized colloidal gold nanoparticles.Furthermore,the characteristics of probes such as stability,antibody coating rate and target capture efficiency were investigated by protein quantification,agarose gel electrophoresis,and stability tests under salt of different concentrations and different pH conditions.The results show that compared to conventional immunocolloidal probes prepared by electrostatic adsorption method,nucleic acid immunocolloidal gold probe has-20%more antibodies binding,is significantly stable at salt concentrations range from 0.5 M to 1 M and pH values among 4-10 as well.The ability of the nucleic acid immunocolloidal gold to bind the target virus is about 40%more higher than that of the traditional immunocolloidal gold.Finally,the nucleic acid colloidal gold probe was used as the probes in lateral flow strip for detection of PRRSV.The preliminary experiment result shows that the PRRSV test strip has a good specificity,repeatability,and stability,and the limit of detection is down to 103 TCID50/mL.