Development And Application Of Hemagglutinin Inhibition Assay And SYBR Green ? Real-time PCR Assay For Detection Of Mycoplasma Synoviae

Mycoplasma synoviae is one of the pathogenic pathogens which causes serious damage to the poultry husbandry,mainly causes chicken or Turkey respiratory diseases and infectious synovitis.In recent years,the infection rate of MS is very high in our country,which has become an important disease restricting the development of poultry industry in China,and it is urgent to develop the rapid diagnosis and detection method of the disease.Isolated culture,ELISA,conventional PCR are Common in domestic diagnosis.But MS ELISA kit is high,the operation time is long,and the price of the enzyme-absorbing instrument is very expensive.Conventional PCR has low sensitivity and false negative possibility,which can only be qualitative and not quantitative detection,the accuracy of the results of MS epidemiological survey was affected.To MS HN01 strains as the research object,this study established the serological level of indirect hemagglutination inhibition(HI)test method of the molecular level of qPCR detection method,it has great significance for the early diagnosis of MS infection,epidemiological investigation and quantitative research.1)First,Establishment of HI assay method of MS.MS HN01 strains from clinical laboratory separation was stationary cultured at 37?to 10~9 CCU/mL.The culture medium was concentrated by centrifugation and the formaldehyde solution was inactivated to prepare diagnostic antigens.Testing the specificity and retention by HI test.Then,MS HN01 antigen plus adjuvant emulsification and other procedures to preparate MS inactivated vaccine.The vaccine was inoculated subcutaneously on the back of a 30-day-old SPF chicken,0.2 mL per one chicken,obtain MS hyperimmune serum by multiple immunizations and test its hemagglutination titer.Finally,the HI test method was used to test the antibody level after inactivated vaccines immunized chickens,compared with the ELISA method.The results showed that,MS HN01strain had no reaction with the pathogenic positive serum of Newcastle disease virus(NDV),Avian infectious bronchitis virus(IBV),Avian influenza virus(AIV),Avian reovirus virus(AAV),Mycoplasma galliscepticum(MG),it showed specificity of the antigen is strong.MS HN01 strains were freeze-dried and save at 20?with 1week,1 month,3 months,6 months,12 months,hemagglutination(HA)titers are unchanged,it showed stability of the antigen is good.Using the HI method to detect four batches of serum,the detection rate was the same as that of the ELISA method,the results show that the established HI method can be initially applied to monitor the immune effect of MS inactivated vaccine.2)Establishment of qPCR detection method of MS.Prepared the positive standard first,our study designed the primers MS-F/MS-R according to the MS vlhA gene sequence collected by GeneBank.MS HN01 genomic DNA as the model,the target gene vlhA(208bp)was amplificated and the recombinant plasmid was cloned in pMD18-T vector.The concentration of recombinant plasmid is determined and the copy number is calculated according to the formula,which is the qPCR positive standard.Then,established the standard curve.the recombinant plasmid was diluted by 10 times the gradient dilution series,SYBR Green?qPCR method was established by optimizing primer concentration and annealing temperature.The sensitivity,specificity and reproducibility of the established qPCR method were tested.Finally,used qPCR and the conventional PCR method to detecte clinical suspected infected chickens internal organization,joint fluid,Claw fluid and chick chorioallantoic membrane(CAM)during the clinical onset of a chicken egg hatching to 9-day age,compared with conventional PCR method.At the same time,the established qPCR method was used to determine the toxic content of MS of different tissues and the detoxification of MS of infected SPF chickens.The results showed that,the concentration of recombinant plasmid DNA was 15.7mg/mL,the copy number was4.95 x 10~122 copy/?L according to the formula.Correlation coefficient R~2 of the established standard curve is 0.999,showing a good linear relationship.Minimum detection limit was 4.95 x 10~2 copy/?L and it's 1000 times higher than normal PCR,it showed that the method had highly sensitive;without cross-reaction to MG,IBV,NDV,AIV and other common avian pathogens,it showed that the method had strong specificity;and a good stability and repeatability while the coefficient of variations are both less than 2%.The qPCR method for the detection of clinical samples was higher than the conventional PCR method.Student t-test statistical analysis showed that the sensitivity of the qPCR method was significantly higher than that of the conventional PCR method(P<0.05).MS artificial infection chick embryo test and SPF chicken infection test results showed that the higher MS content of the CAM and yolk membrane,infected SPF chickens with higher MS content in the throat swab.In this study,the SYBR Green I qPCR provides a rapid quantitative detection technique for early detection and diagnosis of MS.