肺脏组织中绵羊肺炎支原体核酸原位富集方法的建立及应用OA

To address the challenges in genetic evolution and epidemiological studies of Mycoplasma ovipneumoniae(Mo)caused by its remarkable genomic diversity and the time-consuming isolation and culture procedures,sheep suspected of Mo infection in Gansu Province were subjected to necropsy observation and histopathological examination in this study.Clinical samples were examined using real-time fluorescence quantitative polymerase chain reaction(qPCR),and lesion lung tissues without prior Mo nucleic acid enrichment were analyzed by metagenomic next-generation sequencing(mNGS).Subsequently,an in situ nucleic acid enrichment method for Mo in lung tissues was established through a three-factor experiment involving sampling site selection,differential centrifugation enrichment,and enzymatic digestion conditions.Using this method,Mo nucleic acids in lung tissue samples were enriched and directly sequenced for whole-genome determination,followed by genetic variation analysis of the enriched samples.The results showed that Mo predominantly infected the right apical lobe of sheep lungs.Hematoxylin-eosin(H.E.)staining revealed extensive inflammatory cell infiltration in the alveolar septa and interstitium of infected sheep lungs.qPCR detection showed a positive rate of 66.67%(30/45)for Mo.After in situ enrichment treatment,the sequencing output decreased from(16.27±0.62)Gbp to(1.41±0.56)Gbp,while the proportion of Mo sequences increased from(57.4±3.7)×10⁻⁶ to(2 124.7±2 172.7)×10⁻⁶.Genome coverage of Mo increased from(0.99±1.02)%to(91.87±6.28)%,indicating the successful establishment of the in situ enrichment method for Mo nucleic acids in lung tissues.Genetic variation analysis demonstrated that the enriched Mo sequences clustered closely with the domestic isolates NXNK2203,NJ01,and NM2010.These findings indicate that a simple and highly reproducible in situ enrichment method for Mo in lung tissues applicable to mNGS was successfully established,providing technical support for rapid molecular detection and molecular epidemiological studies of Mo.