牛源多杀性巴氏杆菌主要荚膜群和脂多糖基因型两组三重PCR检测方法
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S855.1

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现代农业产业技术体系北京市奶牛创新团队岗位专家项目(BA1C06-2021);国家重点研发计划重点专项(2016YFD050095)


Two Sets of Triplicate PCR Assays Detecting Major Capsular Serogroups and Lipopolysaccharide Genotypes of Bovine Pasteurella multocida
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    摘要:

    为掌握国内牛源多杀性巴氏杆菌流行的主要荚膜群及脂多糖基因型,建立了检测多杀性巴氏杆菌(Pm)及其A、B荚膜群的三重PCR(PmAB-3PCR)以及检测3个脂多糖基因型的三重PCR(LPS-3PCR)方法,检验了其特异性和敏感性,用感染Pm小鼠的组织和人工污染Pm的牛鼻拭子样品进行了临床模拟检验,并对30株Pm进行了PCR检测和传统血清学分型比较。结果显示:PmAB-3PCR特异性好,PmAB-3PCR和LPS-3PCR的DNA检测限分别为10~100 pg和1 ng,二者对菌液的检测限分别为200~2 000 CFU和2 000~20 000 CFU。模拟临床样品PmAB-3PCR检测结果与细菌分离结果100%一致。PmAB-3PCR与琼扩试验的符合率为84%,二者检出的阳性率分别为88%和72%,差异不显著(P>0.05);LPS-3PCR与琼扩试验的符合率为60%,检出的阳性率分别为90%和50%,差异显著(P<0.05)。结果表明,建立的两组三重PCR方法准确高效且重复性好,可取代常规血清学方法用于国内牛巴氏杆菌病的诊断、流调和疫苗株筛选。

    Abstract:

    In order to investigate major capsular serogroups and lipopolysaccharide genotypes of bovine Pasteurella multocida(Pm)in China,two sets of triplicate PCR assays(including PmAB-3PCR and LPS-3PCR)were established to detect Pm and its capsular serogroups A and B as well as three lipopolysaccharide genotypes,respectively,followed by detection of their specificity and sensitivity,then clinical simulation test was conducted with the tissues of mice infected with Pm and bovine nasal swab samples,and 30 Pm strains were detected by PCR and compared with traditional serotyping. The results showed that PmAB-3PCR was with good specificity,the detection limits were determined as 10 to 100 pg DNA or 200 to 2 000 CFU for PmAB-3PCR and 1 ng DNA or 2 000 to 20 000 CFU for LPS-3PCR. The results of the simulated clinical test were 100% consistent with bacteria isolation. The coincidence rate of PmAB-3PCR and agar diffusion test was 84%,and their detection positive rates were 88% and 72% respectively,with not significant difference(P>0.05);whereas the coincidence rate of LPS-3PCR and agar diffusion test was 60%,their detection positive rates were 90% and 50% respectively,with significant difference(P<0.05). In conclusion,the established two assays could be used to diagnose the disease,conduct epidemiological investigation or select appropriate vaccine strains instead of routine serological methods due to its advantages of accuracy,effectiveness and repeatability.

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李霄阳,许建,刘文晓,章振华,徐福洲,陈小玲,李永清.牛源多杀性巴氏杆菌主要荚膜群和脂多糖基因型两组三重PCR检测方法[J].《中国动物检疫》编辑部,2022,39(1):97-104.

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