Establishment Of QPCR And Antibody Immunochromatographic Detection Methods For Mycoplasma Synoviae

Mycoplasma synoviae is a common avian pathogen that causes infection and disease in chickens,mainly infecting chickens or turkeys,causing acute or chronic subclinical respiratory disease and infectious synovitis in chickens,which has caused huge economic losses to the poultry industry in China.Currently,the commonly used diagnostic methods for MS include pathogen isolation and culture,enzyme-linked immunosorbent assay(ELISA),PCR method and so on.However,these methods may be limited by the slow growth,poor sensitivity and non-specific cross-reactivity of the organism in performing large-scale clinical sample testing,so the establishment of a rapid,highly accurate,sensitive,simple and economical test is of great significance for the early diagnosis of MS.In this study,MS qPCR and MS antibody detection by coloured microsphere strips were developed to provide an effective tool for the early clinical diagnosis of MS.Establishment of MS qPCR assay.Firstly,MS positive standards were prepared.Genomic DNA was extracted from MS strains after resuscitation and expansion of culture,and its concentration was measured and copy number was calculated according to the formula for positive standards for qPCR.The genomic DNA was first diluted to 1×10~7and then 10-fold gradient dilution.The primers MS-F/MS-R were designed according to the vlhA gene sequence of MS WVU-1853 strain on Gen Bank,and the standard curve of TaqMan fluorescent qPCR method was established by optimizing the primers,probe concentration and annealing temperature,and the established fluorescent qPCR method was specificity,sensitivity and reproducibility tests.The established fluorescent quantitative PCR method was also used to test pharyngeal swabs and joint fluid swabs from chickens with clinical suspicion of MS infection from a number of farms in Binzhou and breeding eggs during the clinical onset of MS,and compared with the conventional PCR method.The results showed that the established standard curve correlation coefficient value(R~2)was 0.999 and the amplification efficiency(E%)was 96.494,showing a good linear relationship.The method has a minimum detection limit of1×10~0copies/μL,which is higher than that of ordinary PCR,indicating its high sensitivity.The MS genome and common clinical mycoplasma pathogens,such as MG、MHp、Mhr、MO、M.bovis were used as templates for amplification and only MS was amplified,but not for any other pathogen genomes,indicating its high specificity.Three qPCR assays were performed with three concentrations of genomic DNA as templates,and three replicates were performed for each amplification,and their coefficients of variation were less than 3%,indicating good reproducibility.The qPCR method and conventional ordinary PCR in this study were used to test clinical samples simultaneously for comparative analysis,and the results showed that the positive detection rate of qPCR was 89.28%The positive detection rate of conventional PCR was 27.27%,and its sensitivity was significantly higher than that of the conventional PCR method.The experimental results showed that the MS qPCR method established in this study is sensitive,specific and reproducible,providing a rapid quantitative method for the early diagnosis of MS infection.Development of colourful microsphere test strips for MS antibody detection.Firstly,the antigen was prepared,the cultured MS was concentrated,washed and crushed,the soluble protein of MS crushing was taken as the sensitizing antigen and coupled with colouful nano-microspheres,the antibody obtained from New Zealand rabbits with triple immunization of MS was wrapped on the quality control line as the quality control antibody,and the protein of MS crushing was wrapped on the detection line as the labelled antigen,and the colouful microsphere immunochromatographic test strips for MS antibody detection were prepared.A rapid method for the detection of MS antibodies was established by optimising the detection conditions,specificity,sensitivity and stability tests.The results of the specificity test showed that the microspheres sensitized with MS fragmentation protein only reacted with MS positive sera to produce bands,and no bands were produced with positive sera of other common avian pathogens,indicating their specificity.The results of the stability test showed that the sensitized microspheres prepared in different batches and stored at 4℃reacted with the positive sera and all produced bands with good stability.The above shows that the colouful microsphere strips developed in this study have good specificity,sensitivity and stability for the detection of MS antibodies,providing a rapid,simple and economical method for the detection of MS antibodies.In summary,this study successfully established a qPCR assay for MS with high sensitivity,high specificity and good reproducibility,and a colour microsphere test strip assay for MS antibody detection,which is sensitive,specific,rapid and convenient.Both of these rapid diagnostic methods can quickly and effectively detect the presence of MS infection in chickens,providing an effective method for the mass and rapid detection of MS antigens and antibodies.