兔源强毒力金黄色葡萄球菌双重PCR检测方法的建立

A Duplex PCR Assay for Simultaneously Detecting Two Virulent Strains of Staphylococcus aureus in Rabbits

【目的】建立检测兔源强毒力金黄色葡萄球菌(Staphylococcus aureus)的双重PCR检测方法,为兔葡萄球菌病的诊断提供技术支持。【方法】根据兔源金黄色葡萄球菌的nuc和pvl两种毒力基因的保守序列分别设计了两对特异性引物进行双重PCR扩增,对双重PCR反应体系的混合引物浓度和退火温度进行优化,对双重PCR检测方法的特异性、敏感性和准确性进行验证。【结果】当双重PCR反应体系混合引物的浓度为0.6μmol·L^-1、退火温度为59.6℃时,双重PCR的扩增效果最优。该双重PCR检测方法特异性好,能扩增出兔源强毒力金黄色葡萄球菌的nuc(320 bp)和pvl(585 bp)基因片段以及低毒力菌株的nuc(320 bp)基因片段,对兔源多杀性巴氏杆菌、支气管败血波氏杆菌、肺炎克雷伯菌和大肠杆菌等4种常见兔源细菌性病原菌以及阴性对照均无交叉反应。该方法敏感性高,能分别检出10 pg和100 fg的强毒力和低毒力兔源金黄色葡萄球菌基因组DNA。该方法重复性好,批内和批间重复性试验的变异系数均为0;该方法准确性好,对119份临床样品的检测结果与已报道的检测金黄色葡萄球菌nuc和pvl基因的单重PCR检测方法的符合率为100%。【结论】针对nuc和pvl两种毒力基因建立的双重PCR检测方法具有良好的特异性、敏感性、重复性和准确性,为兔源强毒力金黄色葡萄球菌的检测提供了有效的技术支持。

【Objective】A duplex PCR assay for simultaneous detection on hyper-and low-virulent Staphylococcus aureus strains in rabbits was developed.【Methods】The assay was based on the specific primers targeting the nuc and pvl genes in a hyper-virulent and a low-virulent strain of S.aureus isolated from rabbits in Fujian.Primer concentration and annealing temperature for the assay were optimized,and detection specificity,sensitivity,and accuracy determined.【Results】The optimized primer concentration was 0.6μmol·L^-1 and annealing temperature 59.6℃for the assay.Only the hyper-and lowvirulent S.aureus isolated from rabbits were detected with no cross-reactions on the commonly found pathogens,such as Pasteurella multocida,Bordetella bronchiseptica,Klebsiella pneumonia,and Escherichia coli,nor on the negative control.The methodology was highly sensitive with a detection limit of 10 pg genomic DNA on the hyper-virulent strain and 100 fg on the low-virulent strains.The coefficients of variation of intra-and inter-assay were 0 indicating high repeatability on both applications.In addition,the test results of the duplex PCR assay were 100%coincident with those obtained by the PCR assays targeting nuc and pvl individually.【Conclusion】The newly developed duplex PCR assay could simultaneously detect the two virulent S.aureus strains with high specificity,sensibility,repeatability,and accuracy.