Genome Analysis,Construction And Application Of TaqMan-MGB Probe Detection Method Of Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae(Mo)is the main pathogen causing contagious pleuropneumonia in sheep,goats,deer and small ruminants.The disease is a highly contagious infectious disease with rapid onset,wide spread and difficult to eradicate.It has become one of the major diseases that seriously threaten the vigorous development of China ’s sheep industry.Therefore,a rapid,efficient and sensitive Mo detection method is established to provide effective support for the rapid diagnosis and prevention and control of Mo disease.In this study,the whole genome sequence of Mo strain isolated in Ningxia Hui Autonomous Region was determined,and bioinformatics analysis,conserved target gene screening,TaqMan-MGB real-time fluorescence quantitative detection method of Mycoplasma ovipneumoniae were established.The epidemiological investigation of Mycoplasma ovipneumoniae in Ningxia Hui Autonomous Region was carried out by using the established detection method.The results are as follows:(1)The whole genome sequence of Mo NX-16 strain was determined and bioinformatics analysis was performed using SMRT third-generation sequencing technology of PacBio company.The results showed that the total length of NX-16 genome was 1,014,835 bp,and the GC content was 29.23%.The number of predicted genes was 686,the total length of the sequence was 878,010 bp,and the average length was 1,279 bp.A total of 31 signal peptides,211 transmembrane proteins and 20 secretory proteins were predicted.There are at most 95 genes related to translation,ribosomal structure and biological origin,and at least one gene related to signal transduction mechanism.This study will explore the important information of this strain from the perspective of biological information,and provide genes for the subsequent establishment of PCR detection methods.(2)Based on the results of whole genome sequencing,the target gene of Mycoplasma ovipneumoniae Transketolase was screened as a conserved gene.By designing primers and TaqMan-MGB probes for Transketolase gene,constructing standard positive plasmids,establishing standard curves and carrying out specificity,sensitivity and repeatability studies.The results showed that the standard curve of the detection method had a good linear relationship.The correlation coefficient of the standard curve was R2=0.999,the slope was K=-3.131,the amplification was E=104.17%,the intercept was Intercept=40.34,and the regression equation of the standard curve was y=-3.131x+40.34.There was no amplification curve for 8 pathogens such as Mycoplasma bovis,Mycoplasma mycoides subsp.capri,Pasteurella,Bacillus and Salmonella.The minimum detection concentration of the sample was 1×101 copies/μL,which was 100 times higher than that of the ordinary PCR detection method.The coefficient of variation within and between groups was less than 2%,indicating that the Mo TaqMan-MGB probe detection technology established in this study has strong specificity,high sensitivity and good repeatability.The detection method can detect Mycoplasma ovipneumoniae quickly,sensitively and efficiently,and provide technical support for the diagnosis and prevention of the disease.(3)In order to grasp the incidence and epidemic trend of Mo in sheep flocks between different counties/districts in Ningxia Hui Autonomous Region,400 nasal swabs collected from sheep in main sheep breeding counties/districts in 2022 were detected by Mo TaqMan-MGB probe detection technology established in this study.The results showed that 332 samples were positive for Mo,with an average positive rate of 79.25%.The detection rate of Mo in Lingwu County was the highest,which was significantly different from that in other districts and counties(P<0.01).The detection rate of Mo in Helan County was significantly different from that in Yanchi County(P<0.05).The detection rate of Mo was the highest in 3～5 Months old,which was significantly different from that of 2 Months old,2 years old and 3 years old sheep(P<0.01),indicating that Mo infection occurred in different counties/districts in Ningxia Hui Autonomous Region,and the peak period of infection was 3～5 Months old.