禽波氏杆菌16S rRNA和ompA基因双重PCR检测方法的建立

Establishment of Duplex PCR Method Base on 16S rRNA and OmpA Genes for Detection of Bordetella avium

根据GenBank中登录的禽波氏杆菌(B.avium)16S rRNA和ompA基因序列设计引物,经PCR反应条件优化,建立了检测禽波氏杆菌的双重PCR方法。该方法能够同时扩增B.avium基因组中的16S rRNA和ompA基因的特异序列,而对禽源大肠埃希菌(Escherichia coli)、沙门菌(Salmonella)、铜绿假单胞菌(Pseudomonas aeruginosa)等菌株的扩增结果均为阴性。该方法对B.avium基因组DNA检测的敏感性可达1.25×10^4 copies/μL,对其菌液检测的敏感性可达1.2×10^3 cfu/mL,对临床样品的检测结果表明,建立的双重PCR方法与16S rRNA单一PCR检测方法符合率为100%。建立的双重PCR检测方法为B.avium的检测提供了快速、准确、灵敏、特异的检测方法。

In order to accurately and quickly identify Bordetella avium,this experiment designed primers based on the B.avium 16S rRNA and ompA gene sequences recorded in GenBank,and established the duplex PCR method for the detection of B.avium after the optimization of PCR reaction conditions.The results showed that the method can simultaneously amplify the specific sequences of 16S rRNA and ompA gene in B.avium genome,but Escherichia coli,Salmonella,Pseudomonas aeruginosa controlled strain amplification results were negative.The sensitivity of this method to the detection of genomic DNA of Bordetella avium can reach 1.25×10^4 copies/μL,the sensitivity to the detection of germ solution can reach 1.2×10^3cfu/mL,and the detection results of clinical samples showed that the coincidence rate between the established duplex PCR method and the simplex PCR method of 16S rRNA was 100%.The duplex PCR detection method established in this study provided a rapid,accurate,sensitive and specific detection method for the detection of B.avium.