Development And Application Of Fluorescence Quantitative PCR Assay To Detect Mycoplasma Suis

Porcine erythroblastosis is a zoonotic disease that erythrocyte cells are attached to porcine red blood cells,plasma and bone marrow.The main symptoms are fever,anemia,and jaundice.Porcine erythroblastosis often does not occur alone.It is generally mixed infected with PRRS and swine fever.The present situation is very serious and difficult to control.It has caused huge economic losses to the pig industry in China.At present,in the clinical diagnosis of veterinary,the main method is microscopic examination,except blood pressure film method blood smear staining method,but they can not accurately identify pathogens and are easily misdiagnosed.Therefore,in the clinical diagnosis of porcine erythroblastosis,it is urgent to establish an accurate,efficient and specific detection method.This experiment mainly includes three experimental parts: 1.Construction of positive standard plasmid.2.Establishment of a quantitative PCR assay for the detection of erythroblastosis in pigs.3.Clinical application of fluorescent quantitative PCR assay for pig erythrocyte disease.According to the erythrocyte cell adhesion gene MSG1,to design a pair of specific primers the conserved region to amplify the sample DNA,and the gel was recovered and sequenced.The target fragment of the sequencing result was 193 bp,which was consistent with the expected result.A positive plasmid was constructed to use DNA and a standard curve in the quantitative PCR assay for erythropoiesis was established.Fluorescence quantitative PCR detection method of porcine Eperythrozoon was established by positive plasmid,which shows that the amplification curve was S-shaped,and the Ct values between the concentration gradients were relatively uniform.The melting curve proves that a single melting peak indicates no primer dimer production.The Tm value was 83.5°C.The linear equation of the standard curve is Y=?2.6342X+39.855,the slope of the standard curve is ?2.6342,the intercept is 39.855,and the correlation coefficient R2 of the reaction is 0.9977.The method is specific which does not cross-react with healthy pig blood,porcine reproductive and respiratory syndrome virus,chlamydia,lactic acid bacteria,and porcine ring type 2 virus.The coefficient of variation of the repetitive experiments in the group ranged from 0.84% to 2.98%,which was less than 5%.The Ct value between the groups was between 0.650% and 4.078%,which was less than 5%.This result indicates that the method has good repeatability.The lowest concentration of positive plasmid standards was 8.27 x 101 copies/ul.This method's the sensitivity is 1000 times higher than ordinary PCR detection methods.In the same 30 blood samples,the positive rate of real-time PCR detection was 63.3%,and the positive rate of common PCR detection was 50%.The positive detection rate of real-time PCR detection method is 13.3% higher than that the common PCR detection method.The 204 blood samples from Yueyang,Hengyang,Fuyang,and Youxian were tested.The results showed that eperythrozoon infection was found in all the four counties;the infection status of different populations was: sow > Boars > Piglets.Conclusion: This study established a quantitative PCR assay for erythropoiesis disease in pigs.The method has the advantages of high efficiency,accuracy,good specificity,and simple operation.The test results are more objective and direct,and can more accurately determine negative,positive and suspicious samples.Through the detection of samples from different regions,it was found that the infection of erythroblastosis in pigs existed in various regions of Hunan and various growth stages of pigs.