Establishment Of PCR-ELISA Detection Method For Clostridium Anthracis

Clostridium chauvoei is an acute,febrile,septic and highly lethal infectious disease caused by Clostridium chauvoei that primarily infects ruminants such as cattle and sheep.The pathogen is susceptible to invasion of the damaged animal organism,with budding spores invading from the throat or mouth to the blood or injured tissue.The mortality rate is clinically confirmed to be age-dependent,with the older the age,the relatively lower the infection and mortality rates,and with distinct endemic features.Without targeted intervention,disease mortality increases and farmer turnover decreases.Gas gangrene,with insidious pre-symptoms,is an infectious disease with high mortality and low cure rate,which is difficult to prevent and control clinically and is detrimental to the normal development of the livestock industry and thus a threat to human life and health,and is currently receiving high attention worldwide.Therefore,a simple,specific and sensitive PCR-ELISA assay was developed for the epidemiological investigation and diagnosis of C.aeruginosa.Two pairs of primers were designed based on the sequence of C.aeruginosa NagH gene published in GenBank(accession no.KY064176.1)using Primer Premier software,and the non-radioactive primers biotin(Bio)and digoxin(Dig)were labeled at the upstream and downstream 5' ends of one of the primers,respectively.Clostridium perfringens positive samples were used as the study subjects,and the genomic DNA was extracted and PCR amplified,and the target gene fragments were obtained and purified for recovery.It was cloned into pMD-19T simple vector,plasmid PCR followed by double digestion,and the positive results obtained were sent to Bio for testing.The PCR product dilution,closure time,streptavidin concentration,PCR product dilution,HRP-anti-digoxin antibody color development time and dilution were optimized to select the most suitable reaction conditions and operated according to the PCR-ELISA procedure.Thirty-five negative C.aeruginosa samples were selected for PCR-ELISA after PCR testing,and the OD470 nm value was measured to calculate the threshold value for C.aeruginosa PCR-ELISA and select more accurate judgment criteria.The next fold ratio dilution of C.aeruginosa genomic DNA was applied to the PCR-ELISA assay to determine its sensitivity.Suitable reaction conditions were selected,and genomic DNA of Pasteurella,Clostridium perfringens,and Clostridium perfringens types D and E were used as reaction templates to determine the specificity of the PCR-ELISA assay.One hundred and fifty bovine anticoagulated blood samples were tested for the stability of the repeatability of the enzyme-coated plates at different times and at the same time,and the stability of the assay was compared with that of the conventional method.The results showed that the band size after conventional PCR was about 1 107 bp,which was 99%homology with the sequence of C.aeruginosa gene[KY064176.1]published on Genbank,proving that the amplified target fragment was C.aeruginosa.After optimizing all aspects of the experiment,the following conclusions were drawn:the critical value for C.aeruginosa negative samples was 0.174,with negative meaning that samples with OD470nm values ?0.234 and vice versa were positive.The appropriate blocking time was 60 min,the appropriate substrate development time was 15 min,the appropriate dilution of PCR product and HRP-labeled anti-digoxin antibody was 30x and 2000x,respectively,and the appropriate concentration of streptavidin was 5 ?g/mL.The lowest concentration of C.aeruginosa DNA detected by PCR-ELISA was 2.2 pg/?L The sensitivity was 100-fold higher than the 220 pg/?L detected by PCR,and there was no cross-reactivity with Clostridium perfringens type D,Clostridium perfringens type E,Clostridium perfringens,and Pasteurella spp.with strong specificity.The positive detection rate of 150 anticoagulated bovine blood was 21.3%,which was higher than that of 16.0%by conventional PCR.