Evaluation Of Serological Diagnosis Of Hsp 70 And EF-Tu In Mycoplasma Ovipneumoniae And Molecular Diagnostic Study Based On LAMP

Objective:?1?Develop a DNA purification and separation method that is simple,rapid,and effective for the substrate.?2?Establish a simple and rapid detection technique for M.ovipneumoniae nucleic acid amplification.?3?To solve the control and prevention problem of M.ovipneumoniae infection,explore and establish a simple,rapid and economical M.ovipneumoniae detection method suitable for the grassroots.Methods:?1?Preparation of superparamagnetic Fe₃O₄ MCNCs and Fe₃O₄/SiO₂ MCNCs,and extraction of DNA from samples of different sources by MCNCs,including rabbit whole blood,rabbit frozen blood and lung tissue infected with M.ovipneumoniae,verify its purity,integrity and yield.?2?Design a set of specific primers for M.ovipneumoniae heat shock protein 70?Hsp 70?and elongation factor Tu?EF-Tu?genes,and use nucleic acid amplification by loop-mediated isothermal amplification?LAMP?technique.The lateral flow test strip technique?LFD?enables visual detection of nucleic acid amplification,establishes a rapid detection method of M.ovipneumoniae LAMP-LFD,and detects its specificity,sensitivity and clinical application.?3?Construction of prokaryotic expression vector of M.ovipneumoniae EF-Tu and Hsp 70 genes,induction of purified protein and analysis of its immunogenicity by Western-blot method,and establishment of M.ovipneumoniae indirect by using Hsp 70 and EF-Tu proteins as diagnostic antigens.The ELISA method was used and its specificity and sensitivity were assessed.Results:?1?Fe₃O₄ MCNCs with particle size of 150-200 nm were prepared.The particle size of Fe₃O₄/SiO₂ MCNCs was 195-300 nm,the thickness of SiO₂ was about 25 nm,and the SiO₂ layer was completely wrapped with Fe₃O₄ MCNCs.Dispersed state;VSM detected Fe₃O₄ MCNCs saturation magnetization?Ms?was 46.2 emu/g,Fe₃O₄/SiO₂ MCNCs measured saturation magnetization?Ms?was33.6 emu/g.The DNA bands extracted by MCNCs in each sample were intact,with no obvious rupture,good concentration and purity,and high extraction efficiency.?2?Hsp 70 gene LAMP can specifically detect the presence of M.ovipneumoniae by reacting at 62?for 70 min;EF-Tu gene LAMP can be detected specifically at 62?for 80 min to detect the presence of M.ovipneumoniae.The total coincidence rate of M.ovipneumoniae Hsp 70 LAMP-LFD detection method and pathogenic detection method was94%;and the total coincidence rate of established M.ovipneumoniae EF-Tu LAMP-LFD detection method and pathogenic detection method was 80%,indicating The sensitivity of the established M.ovipneumoniae Hsp 70 LAMP-LFD assay and the coincidence rate with the pathogen detection method were higher than those of the M.ovipneumoniae EF-Tu LAMP-LFD assay.?3?The optimized EF-Tu indirect ELISA reaction conditions showed that the antigen dilution was 5?g/mL,the optimal dilution of the primary antibody was 1:50,the optimal incubation time was 90 min,and the blocking solution was 5%skim milk powder.The optimal dilution ratio of the secondary antibody is 1:8 000,the optimal incubation time is 90min,and the specificity and sensitivity are good.The method and the indirect hemagglutination method are used to detect 100 serums,and the coincidence rate is 81%;Optimization of Hsp 70 indirect ELISA reaction conditions showed that the antigen dilution was 0.25?g/mL,the optimal dilution of the primary antibody was 1:50,the optimal incubation time was 90 min,the blocking solution was 5%skim milk powder,and the secondary antibody was optimally diluted.The multiple is 1:8 000,the optimal incubation time is 60 min,and the specificity and sensitivity are good.The method and the indirect hemagglutination method are used to detect 100 serums,and the coincidence rate is 90%.Conclusions:?1?The method for the isolation and purification of nucleic acids based on MCNCs is simple and rapid,without the use of proteinase K or toxic compounds,for the automatic extraction of nucleic acids in the future,and also provides an effective means for the rapid extraction of nucleic acids in the grassland farming and pastoral areas.?2?The LAMP-LFD method established in this study has the advantages of high specificity,high sensitivity and simple and convenient operation,which provides technical support for the prevention and diagnosis of M.ovipneumoniae.?3?The established M.ovipneumoniae indirect ELISA method provides a rapid and effective screening method for M.ovipneumoniae serological testing,and can also be widely used.