Accurate Detection Of Streptococcus Pneumoniae By Using Ribosomal Protein L7/L12 As Molecular Marker

Streptococcus pneumoniae(Sp)is an important pathogen of respiratory infections in children.It is one of the most common high morbidity and mortality bacterial pathogens in the world and can cause a variety of infectious diseases.The current diagnostic methods for detecting Sp infections often cannot meet the needs of rapid clinical detection,long operation cycles,strong professionalism,and false positives,which are greatly limited in practical applications.Given the serious consequences of Sp infections and the shortcomings of current detection methods,it is important to develop methods for accurate and effective detection of Sp.In this study,the ribosomal protein L7/L12 was used as a detection marker,and a method for rapid detection of Sp by colloidal gold immunochromatography was explored.In this study,the gene sequence of ribosomal protein L7/L12 derived from Sp was analyzed at first,and specific primers were designed and synthesized to construct p ET-21a-Sp-RP-L7/L12 recombinant plasmid.After induction of expression,it was purified by affinity purification on a nickel column to obtain a recombinant protein with a purity of more than 90%.Recombinant protein antigens were immunized to healthy female BALB/c mice multiple times.The chemical fusion method was used to fuse the splenocytes of the immunized mice with myeloma cells sp2/0.After subcloning,the hybridoma cell supernatants were screened by indirect ELISA and BLI technology and verified by ascites spots.Two monoclonal antibodies that could specifically react with natural proteins were finally obtained and named Sp-1 # and Sp-2 #.The purified monoclonal antibodies were identified and analyzed by indirect ELISA,Western blot and BLI detection techniques.Indirect ELISA assay,the titer of Sp-1 # antibody is 1: 256000,and the titer of Sp-2 # antibody is 1: 512000.The two antibodies only reacted positively with Sp,but did not cross-react with the other nine common respiratory pathogens.Both antibodies have good affinity,and the antibodies bind to different epitopes of the antigen.They have no competitive effect and can specifically recognize ribosomal proteins L7/L12 derived from Sp.A colloidal gold immunochromatographic test strip was developed based on the principle of double antibody sandwich,and its specificity,sensitivity,and stability were evaluated.Pairing and detection were made on colloidal gold immunochromatography platform,with Sp-1 # as the gold standard antibody and Sp-2# as the detection antibody,the minimum detection limit of the produced test strip is1.0 × 105 CFU / m L.It had a specific reaction with Sp,but did not cross-react with other 9 kinds of common respiratory pathogens such as Hi and Mc.Test strips had good repeatability and stability when stored at 45 ℃ for 37 days.RP-L7/L12 can be used as a detection marker for Sp.The test strips prepared by its monoclonal antibody can be used for rapid detection of Sp.