兔源F型多杀性巴氏杆菌双重PCR检测方法的建立

Duplex PCR Detection on Pasteurella multocida Serogroup F Strain in Rabbits

【目的】建立检测兔源F型多杀性巴氏杆菌的双重PCR检测方法,为兔巴氏杆菌病的诊断提供技术支持。【方法】根据多杀性巴氏杆菌kmt1基因和F型多杀性巴氏杆菌fcbD基因的保守序列分别设计了2对引物进行双重PCR扩增,对双重PCR检测方法的退火温度和混合引物浓度进行优化,并对方法的特异性、敏感性、重复性和准确性进行验证。【结果】该双重PCR方法最优反应条件为:退火温度60℃、混合引物浓度0.8μmol·L^(−1)。该双重PCR方法能扩增出兔源F型多杀性巴氏杆菌的kmt1基因片段(260 bp)和fcbD基因片段(490 bp)、兔源A和D型多杀性巴氏杆菌的kmt1基因片段(260 bp),对兔源支气管败血波氏杆菌、肺炎克雷伯菌、大肠杆菌、金黄色葡萄球菌和阴性对照均为阴性,无交叉反应,特异性较强。该方法对兔源F型多杀性巴氏杆菌的最低检出限为1×10^(3)拷贝·μL^(−1),敏感性高。应用该方法对90份病死兔肺脏样品进行批内和批间重复性试验,结果均一致,重复性好。应用该双重PCR方法和已报道的多重PCR方法同时检测87份已知结果的呼吸道病死兔肺脏样品,结果显示双重PCR方法检测结果和已报道的多重PCR方法检测结果与已知结果的符合率分别为97.70%和94.25%,双重PCR方法检测结果与已报道的多重PCR方法检测结果的符合率为93.10%,双重PCR方法的准确性更高。【结论】针对多杀性巴氏杆菌kmt1基因和F型多杀性巴氏杆菌fcbD基因建立的双重PCR检测方法具有良好的特异性、敏感性、重复性和准确性,为兔源F型多杀性巴氏杆菌的快速检测提供了技术支撑。

【Objective】A duplex PCR assay for rapid detecting Pasteurella multocida serogroup F strain in rabbits was developed.【Methods】Primer concentration and annealing temperature for the duplex PCR were optimized based upon two sets of specific primers targeting the conserved sequences of kmt1 of P.multocida and fcbD of P.multocida serogroup F strain.Specificity,sensitivity,and accuracy of the methodology were tested for its applicability.【Results】The optimized conditions for the assay included 0.8μmol·L^(−1) on the primer concentration and 60℃for the annealing temperature.The assay detection was specific for P.multocida serogroup A,D,and F strains and free of cross-reactions with Bordetella bronchiseptica,Klebsiella pneumonia,Escherichia coli,Staphylococcus aureus,and negative control.A highly sensitive detection limit up to 1×10^(3) copies·μL^(−1) on genomic DNA of P.multocida serogroup F isolate was achieved.In the repeated intra-and inter-assays on 90 lung specimens from dead diseased rabbits,the assay variation coefficients were identical.By using 87 lung samples collected from known infection cases,the newly developed duplex PCR assay and reported multiplex PCR assay yield agreements of 97.70%and 94.25%with the known results,respectively.Moreover,the current duplex PCR determination also yielded an agreement of 93.10%on the measurements with the reported from a multiplex PCR assay.【Conclusion】The newly established duplex PCR assay based on the specific primers targeting the conserved sequences of kmt1 of P.multocida as well as fcbD of P.multocida serogroup F strain was tested to be highly specific,sensitive,repeatable,and accurate in detecting the pathogenic P.multocida serogroup F strain in rabbits.