牛支原体可视化重组酶聚合酶扩增检测方法的建立

Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

为建立一种对牛支原体高效、快捷的可视化重组酶聚合酶扩增(LFD-RPA)临床诊断方法,以牛支原体uvrc基因序列为靶基因,设计特异性引物和探针,通过筛选引物和探针、优化反应条件,并通过灵敏度、特异性、重复性和临床样本检测试验进行验证。结果显示,该试验建立的牛支原体LFD-RPA最佳引物为F2/R2,最佳反应条件为39℃、 25 min;检测灵敏度可达2.08 copies·μL^(-1),是普通PCR灵敏度的100倍;与滑液囊支原体、沙门氏菌、绵羊肺炎支原体、多杀性巴氏杆菌、金黄色葡萄球菌、无乳链球菌和产气荚膜梭菌之间无交叉反应;重复性稳定;对50份鼻拭子样本进行检测,阳性率为26%,与我国行业标准PCR检测方法的符合率为89.6%。该试验成功建立了牛支原体LFD-RPA检测方法,该方法具有操作简便、快速、高效、敏感等优点,为牛支原体的临床快速诊断提供了技术支撑。

The aim was to establish an efficient and rapid visual recombinase polymerase amplification(LFD-RPA)clinical diagnostic method for Mycoplasma bovis.With the uvrc gene sequence of Mycoplasma bovis as the target gene,specific primers and probes are designed and validated by sensitivity,specificity,repeatability and clinical sample detection test through screening of primers and probes and optimizing the reaction conditions.The results showed that the optimal primers for LFD-RPA of Mycoplasma bovis established in this test were F2/R2,and the optimal reaction condition was 39℃for 25 min;the detection sensitivity was up to 2.08 copies·μL^(-1),100 times of common PCR;there was no cross reaction with Mycoplasma synoviae,Salmonella,Mycoplasma ovipneumoniae,Pasteurella multocida,Staphylococcus aureus,Streptococcus agalactiae and Clostridium perfringens;the repeatability was stable;50 nasal swab samples were detected,the positive rate was 26%,and the coincidence rate with domestic industry standard PCR detection methods was 89.6%.In this study,the LFD-RPA detection method for Mycoplasma bovis was successfully established,which has the advantages of easy operation,rapidity,high efficiency,and sensitivity,and provides technical support for the rapid clinical diagnosis of Mycoplasma bovis.