Rapid Detection Of Rotavirus Based On Raman Immunochromatography

Objective:Rotavirus is a double-stranded RNA virus that can infect pigs,cattle,sheep and other animals,causing severe diarrhea and the death of a large number of livestock and poultry,causing serious seconomic losses.Rotavirus also infect humans,and rotavirus diarrhea is one of the four most common diseases in infants and young children worldwide.About 10 million infants and young children are infected with rotavirus every year.This study combines the surface enhanced Raman spectroscopy(SERS)technique with immunochromatography to establish a highly sensitivitive and specific method for the rapid qualitative and quantitative detection of rotavirus.Methods:(1)Preparation of Raman tags:First,gold nanoparticles(Au NPs)were synthesized by sodium citrate reduction method,and the Raman molecule5,5’-Dithiobis-(2-nitrobenzoic acid)was covalently linked to Au NPs through a gold-sulfur bond to prepare Au/DTNB NPs with single-layer DTNB Raman molecules.Silver nitrate was reduced by sodium citrate to create a layer of silver shell on the surface of Au/DTNB NPs to obtain Au/DTNB@Ag NPs.The second layer of DTNB raman molecule were covalently linked to the surface of the silver shell via silver-sulfur bond to form the final raman tags of Au/DTNB@Ag/DTNB NPs.Au/DTNB@Ag/DTNB NPs of series size and various silver shell thickness were prepared by adjusting the concentration of reducing agent sodium citrate in the reaction system and the best Raman tags were screened accorting to the Raman signal strength,partical stability and uniformity.(2)Preparation of Raman tag-antibody conjugated tag(SERS detection tag):The carboxyl groups of DTNB molecules on the surface of Au/DTNB@Ag/DTNB NPs Raman tag were firstly activated by EDC and NHS and then conjugated with the amino groups of the mouse anti-rotavirus antibody 1(Anti-RV m Ab1)via a condensation reaction to obtain the SERS detection labels(SERS-RV m Ab1).The coupling raction was optimized by changing the Raman tag to antibody ratio,activator concentration,blocking time and other conditions according to their stability,uniformity and Raman signal intensity.(3)Development of the rotavirus Raman immunochromatographic test strip:goat anti-mouse Ig G polyclonal antibody and mouse anti-rotavirus monoclone antibody were coated on a nitrocellulose membrane(NC membrane)using a contact-type automatic scoring instrument as the quality control line(C line)and the detection line(T line),respectively.The prepared rotavirus SERS detection probe was coated on the conjugated pad.Then the sample pad,conjugated pad,NC membrane and the absorbent paper are stick on the PVC bottom paper in sequence,and then cut into test strips with a width of 3mm on an automatic cutting machine to obtain the rotavirus Raman immunochromatographic test strip.The coating conditions of antibodises on the NC membrane,the coating conditions of the SERS detection tags on the conjugated pad,the composition of the sample processing solution,and the loading sample volume are optimized.Finally,the specificity,sensitivity and stability of the rotavirus Raman immunochromatographic test strip were evaluated and testing results of clinic samples were compared with those from fluorescent quantitative PCR.Results:(1)Determined the optimal process for the preparation of Raman labeling material was determined:when the average particle size of the gold core was 25 nm,and the modification concentration and time for each layer of Raman molecules was 10 m M and 4 h,respectively,the obtained double-layer Raman molecule labeled gold core-silver shell nanomaterials exhibited good uniformity,high stability and the strongest Raman signal;(2)Optimized the preparation method of SERS detection tags:The optimal conditions for SERS detection tag preparation were found to be a ratio of 500:1 for Au/DTNB@Ag/DTNB NPs to Anti-Rotavirus m Ab1,10m M of activator EDC and NHS,blockage of active sides with 10%BSA for 1h,and 2×gold standard dilution as reconstitution.Under the optimal conditions,SERS detection tags showed the best stability and uniformity,and the strongest Raman signal.(3)Assembled the Raman immunochromatographic test strip for rotavirus detection:The best performance for the test strip were obtained when the T-line and C-line were coated with Anti-RV m Ab2 and goat anti-mouse Ig G in concentrations of 0.7mg/m Land 0.5mg/m L,the conjugated pad was sprayed with 400μL of SERS detection tags,and PBS(p H7.4)containing 0.15%of Tween-20 was used as sample dilution.Under these conditions,the background of the test strip is clean,the color of T and C line is clear,no non-specific T line apprear when negative sample is tested,and the Raman signal of T/C line is strong.The minimum detection limit of the developed test strip for rotavirus in is 8pg/m L,and a linear range were obtained between4pg/m L-4×10~4pg/m L,R~2=0.995.The test strip does not react with syncytial virus,adenovirus,pseudorabies virus and other pathogens,showing good specificity.The coefficient of variation(CV)for the intra-and inter-batch repeated tests were 9%and 3%,respectively.The coincidence rate of the detection results for 85 rotavirus positive stool samples and 10 negative samples was 100%when comparing with the real time quantitative PCR detection(RT-q PCR).Conclusion:A Raman immunochromatographic test was successfully established for the specific,sensitive,rapid,qualitative and quantitative detection of rotavirus The established method can be used for in livestock and poultry farms,grassroots and hospital,exhibiting great significance for the rapid diagnosis,treatment and control of rotavirus diarrhea.