| Porcine epidemic diarrhea virus(PEDV),a coronavirus that causes highly infectious intestinal diarrhea in piglets,has led to severe economic losses worldwide.Rapid diagnosis and timely supervision,which mostly rely on in-lab detection,are of crucial in the prophylaxis of PEDV.Currently,virus-isolation,immunohistochemistry,serological techniques and molecular biological detection methods are most used.However,none of these technologies meet all three requirements:time-saving,highly sensitive,and ease of use.For example,fluorescence quantitative PCR(qPCR)provides high sensitivity,but it needs professional technicians and clean environment.Dark field microscope(DFM)based detection technology,which only needs a common biology microscopy with a concentrator,is available and can be applied for on-site detection of pathogens.Here,we proposed a gold-nanorod(GNR)probeassisted counting method using DFM,the sensitivity of which is comparable to that of qPCR.This work includes the following three parts:1.Preparation and identification of anti-PEDV S1 polyclonal antibody:PEDV S1 gene was obtained through PCR amplification and was inserted into certain location of the pET-28a(+)prokaryotic expression vector.The recombinant plasmid was successfully constructed,and the recombinant protein was obtained through transformation,induction expression and purification.The as prepared recombinant protein was used as immunogen to immunize BALB/c mice and immune serum was then obtained.By indirect ELISA,the serum titer was about 1:12,800,and the specificity of purified antibody was verified by IFA assay and indirect ELISA.2.Preparation of GNR probe and antibody functionalized chip:(1)By combining different concentrations of antibodies with GNRs,the optimal ratio of GNR and antibody on the probe was determined by SDS-PAGE electrophoresis.Subsequently,the probe was characterized by hydrodynamic dimensions,Zeta potential and transmission electron microscope(TEM).Results showed that the probe was successfully prepared with good dispersion and specificity.(2)Polyclonal antibodies were coupled to the silicon wafer by chemical modification.Subsequently,antibodies modified chips that can specifically bind to PEDV were successfully prepared and verified by scanning electron microscopy(SEM).The results showed strong specificity and feasibility of the antibody functionalized chips to capture the virus and form a sandwich structure with GNR probes.3.Construction of quantitative detection of PEDV with GNR probe-based DFM:(1)A qPCR detection system was successfully built to compare with the dark field detection method constructed in this study.Recombinant plasmid which was inserted into N gene was set as the standard product of qPCR,and recombinant plasmid with known copy number was diluted 10-fold gradient as template.Standard curve equation of the qPCR detection system is obtained by calculating the correlation between concentrations and Ct values.(2)The PEDVs in the sample were captured by antibody functionalized chips,and then GNR probes were added to combine with PEDV on the chips to form sandwich structures.Due to the strong scattering characteristics of GNRs,they can be observed and counted with naked eyes under the dark field microscope.The virus samples were gradient diluted by PBS,and then were counted by dark field detection system.The standard curve of dark field counting vs concentration linear relationship was established.Finally,a simulated sample diluted by mouse serum was also detected.The results of samples by GNR probe-based DFM with the limit of detection(LOD)of 23.80 copies/μL are highly consistent with the data by qPCR,indicating that the method we built was reliable for detection of PEDVs in real samples.In summary,the DFM-based quantitative detection strategy constructed in this study had several advantages,including low-cost,time-saving,strong specificity,good repeatability,and,most importantly,could realize field detection of pathogens. |
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