Development Of A PCR-ELISA Assay For The Detection Of Fusobacterium Nucleatum

Fusobacterium nucleatum(F.ucleatum)is a gram-negative anaerobic bacterium,which is also considered as a conditional pathogen.F.ucleatum mainly promotes the occurrence of colorectal cancer(CRC)through the formation of inflammatory environment、immunosuppression、immune avoidance and other pathways.In recent years,scientists have used it as a marker of CRC.Currently,traditional PCR and cycloisothermal amplification are commonly used in the detection of F.ucleatum.These methods have the limitation of low sensitivity and cumbersome operation,so it is necessary to establish convenient and accurate detection methods.This paper achieved this goal through the experimental study of PCR-ELISA and provided technical support for the early detection of CRC.Methods: A pair of specific primers were designed according to the conserved region of F.ucleatum adhesive protein A(Fad A)gene sequence in Gen Bank,the target gene fragment was amplified by PCR,cloned into the pmd-18 vector,the recombinant plasmid was constructed.The target gene fragment was sequenced to prove that the target gene fragment was inserted correctly.Digoxin and biotin were labeled in the upstream and downstream of the specific primer.Using the constructed recombinant plasmid as the template,the target gene was amplified by PCR with marker primers.The reaction conditions of PCR-ELISA were optimized to determine the best reaction conditions.Thirty samples identified as negative by PCR were tested by PCR-ELISA,and the critical value of this method was determined by formula correction = mean value + 3×standard variance(SD).The sensitivity of the template was determined by dilution of the template by 10 times gradient.The specificity of the method was determined by PCR-ELISA for e.coli,salmonella,clostridium botulinum,staphylococcus aureus,clostridium perfringens and Fusobacterium nucleatum DNA to verify their specificity.The repeatability and stability of the method were determined by repeated tests in and between batches of the plate coated at the same time and the plate coated at different times.Fluorescence quantitative PCR primers were designed,recombinant plasmid target gene fragments were amplified,standard curves were established,and fluorescence quantitative PCR methods were constructed.PCR-ELISA was compared with PCR,fluorescence quantitative PCR and bacterial culture identification methods to analyze the accuracyand convenience of the detection method.The results showed that the normal PCR amplification band size was 232 bp,and after sequencing analysis,its homology with the Fad A gene published in Gen Bank was 100%.Through the optimization of each condition of PCR-ELISA,it was finally determined that the optimal package concentration of streptavidin was 1:800(1mg/m L)and placed at 37℃ for 15 min,the concentration of the blocking solution was 2% and placed at 37℃ for 15 min,the PCR product was diluted at 1:50 and placed at 37℃for 30 min,the enzyme labeled secondary antibody was diluted at 1:2000 and placed at 37℃ for 30 min,and the final color was developed for 7min.The 30 samples identified as negative by PCR were tested by PCR-ELISA,and the positive samples were determined to be greater than 0.111 by the formula,while the negative samples were determined to be negative.The PCR-ELISA method established in this study could detect at least 5.86×104copies/μL,while ordinary PCR could detect 5.86×106copies/ μL.The results showed that this method was 100 times more sensitive than ordinary PCR,and had no cross-reaction with escherichia coli,salmonella,clostridium botulinum,staphylococcus aureus,and clostridium perpod,with high specificity.By three clinical feces samples and tested a tissue strain,PCR and fluorescence quantitative PCR detection results for the three feces are negative,an organization is positive.This is consistent with the PCR-ELISA test results.By bacterial culture,the result is also so.This shows that the established PCR-ELISA can do working.Through the comparison of several detection methods,the results showed that pcr-elisa had the advantages of strong specificity,short time,low cost,simple operation and 100 times the sensitivity of PCR.In conclusion,this study established an efficient,fast,specific and sensitive method for the detection of F.ucleatum.It provides technical support for the research and development of detection kits,and provides a fast method for the investigation and early detection of CRC.