Isolation And Identification Of Mycoplasma Bovis And Developments Rapid Diagnosis Methods

M.bovis is a member of Mycoplasmataceae in the class Mollicutes.The genome of M.bovis is 1080 kb and its G+C ratio is 27.8-32.9%.Mycoplasmas form coccoid cells is 0.2-0.3?m in diameter,which cannot be clearly seen by routine light microscopy.Escherichia coli genome is 5 times than mycoplasma.Cattle are infected byM.bovis,M.bovirhinis,M.bovigenitalium,M.bovoculi,M.califomicum,M.canadense,M.dispar,Ureaplasma,M.mycoides,M.agalactiae,M.alkalescens,M.arginini,M.Richteri.Among them,M.bovis,M.califomicum,M.canadense,M.agalactiae and M.alkalescens can cause bovine mastitis;M.bovis,M.dispar,Ureaplasma and M.mycoides can cause bovine pneumonia.M.bovis is a pathogen that mainly caused respiratory disease,mastitis,and arthritis in cattle.The pathogen is widespread in the world and caused great economic losses to the cattle industry.In China,the first M.bovis pneumonia case was reported in Hubei province in 2008.Until now,M.bovis pneumonia cases have been reported in more than ten Chinese provinces.The significance of respiratory tract infection by M.bovis has become increasing in recent years.The main methods of diagnosis mycoplasma are pathogen isolation and identification,serological testing,genetic testing.Serological detection is one of the effective methods and antigen coated ELISA detection method is the main method.At present,indirect ELISA method for detection M.bovis mainly uses the whole protein or recombinant protein coated antigen.At present,there is no commercial ELISA test kit in china.The qualitative and quantitative analyses of original template have come true by real time fluorescent quantitative polymerase chain reaction.Compared with common PCR,the sensitivity,specificity and repeatability of FQ-PCR has improved.Loop-mediated isothermal amplification is a method by using 60-65?temperature conditions amplification gene.The method is convenient,rapid,accurate and low-cost.The sensitivity and specificity of LAMP no less than PCR technology,the cost is lower than Real-time PCR.It is widely used in disease diagnosis,detection and other fields.Objective:M.bovis were studied by the amplification and comparison of some genes in TL2016.Development and application of indirect ELISA for M.bovis antibody.Development and evaluation of real-PCR for M.bovis nucleic acid detection.Development and evaluation of visual detection for M.bovis nucleic acid with LAMP method.Contents:1.To understand the difference between different strains,some genes of TL2016was amplified and alignment.The drug susceptibility of the strain was test.2.Development and application of indirect ELISA for M.bovis.3.Development and evaluation of Real-time PCR for M.bovis nucleic acid detection.4.Development and evaluation for M.bovis nucleic acid with LAMP method.Methods:1.Isolation and identification of mycoplasmaThe pathogen TL2016 was isolated from cattle lung disease which collected in tongliao area.The 16s rRNA,P48,P81 gene was cloned,sequenced and alignment for comparing the differences of different isolates about M.bovis.The drug susceptibility of the strain was test for clinical diagnosis and treatment.2.Development and application of indirect ELISA for M.bovisThe main antigen sites of P48 proteins was analysis by bioinformatics software,then optimized and synthesis P48 genes,amplified P48 main antigen protein,cloning in the pET-32a?+?.P48 total protein and the main antigen protein was expressed,identified and purificated.Indirect ELISA detection method for M.bovis antibody was development and clinical application.3.Development Real-time PCR for M.bovis nucleic acid detectionThrough the analysis of the genome sequence of M.bovis P81,a specific region was screened and a pair of FQ-PCR primers were designed and synthesized in this region.pMD18-T-P81 plasmid was the template of real-time PCR and dilutied into10⁹,10⁸,10⁷,10⁶,10⁵,10⁴,10³,10²,10 copies/?L standard products.The established real-time PCR method was performed specificity,sensitivity and reproducibility.4.Development and evaluation for M.bovis nucleic acid with LAMP methodTwo pairs of specific primers were designed according to six different regions of P81 gene belong 71 bp-296 bp.LAMP method was established to detect M.bovis after optimized the reaction conditions.The specific,sensitive and clinical sample experiments were carried out to analyze the effect of this method.Results:1.In 2016,A strain of M.bovis was isolated from 5 diseased cattle lungs,named TL2016.The 16S rRNA,P81 and P48 genes of TL2016 strains were amplified and alignment.The genetic homogeneity of 16S rRNA betweent different M.bovis is99.9%.The homology of P81 gene in domestic strain is 99.9%.P48 genes is highly conserved and homology.The drug susceptibility test found TL2016 was very sensitive to kanamycin,levofloxacin,dazycin,ciprofloxacin,and ofloxacin;Highly sensitive to tetracycline and chloramphenicol;resistant to bramycin,streptomycin,polymyxin,gentamicin,amikacin and erythromycin.2.The prokaryotic expression vector pET-32a-P48?28-185?,pET-32a-P48?221-455?,pET-32a-P48 was constructed successfully and all expressed the target protein in large quantities.The purified recombinant protein was identified by western blot and the sizes of them were 32kDa,39kDa and 60kDa respectively.P48?221-455?protein was more sensitive than P48?28-185?and P48 protein.ELISA optimum working condition:5%horse serum blocking 1h,serum incubation time 1h,HRP labeled rabbit anti-cow IgG dilution ratio of 1:2000.TMB room temperature display color 15 min,2 M H₂SO₄ ter mination reaction.3.According to the P81 gene of M.bovis,a real-time PCR detection method was established.The minimum detectable concentration of real-time PCR was 10copies/?L,1000 times higher than normal PCR.The correlation coefficient of the standard curve is 0.995.The coefficient of variation was less than 1.5%,with good accuracy and repeatability.The established real-time PCR was suitable for clinical testing.4.According to the P81 gene of M.bovis,a LAMP detection method was established.The minimum detectable concentration of LAMP was 10³ copies/?L,10times higher than normal PCR.LAMP optimum reaction conditions:Bst polymerase0.20?L in 25?L system,64?amplification,amplification time 40 min.The results showed that the change of magnesium and betaine concentration had little effect on the results.The established LAMP method was high specificity and sensitivity.Conclusion:1.A strain of M.bovis was isolated and named TL2016.The genetic homogeneity of 16S rRNA between different M.bovis is 99.9%.The homology of P81 gene is99.9%-94.8%.P48 genes is highly conserved and homology.The drug susceptibility test found TL2016 was very sensitive to kanamycin,levofloxacin and so on.2.The pET-32a-P48?28-185?,pET-32a-P48?221-455?,pET-32a-P48 was constructed,expressed,purified successfully.P48?221-455?protein was more sensitive than P48?28-185?and P48 protein,so the ELISA detection method was established by P48?221-455?protein.This method had a good reiteration,a sharp specificity and a high sensibility,it also has a high coincidence rate with commercial kit.3.According to the P81 gene of M.bovis,a real-time PCR detection method was established.The minimum detectable concentration of real-time PCR was higher than normal PCR and has good accuracy and repeatability.The established real-time PCR was suitable for clinical testing.4.According to the P81 gene of M.bovis,a LAMP detection method was established.The minimum detectable concentration of LAMP was higher than normal PCR.The established LAMP method was high specificity and sensitivity.