禽类三种常见病原菌快速检测体系的建立及应用

Establishment and Application of a Rapid Detection System for Three Common Bacterial Pathogens in Birds

鸭疫里默氏杆菌、大肠杆菌和沙门氏菌是禽类3种常见的革兰氏阴性致病菌。为实现3种病原菌的快速检测,选择各自的保守基因ompA、phoA和fimW,建立并优化了同时鉴定3种病原菌的多重PCR方法。3个引物对的最佳终浓度分别为0.60、0.28、0.20μmol/L,最佳退火温度为57℃;该方法能从其他7种临床常见病原菌中特异性区分出鸭疫里默氏杆菌、大肠杆菌和沙门氏菌;在DNA水平和菌水平的最低检测限分别达到1 pg/μL和10^(4)cfu/mL。本试验建立的多重PCR方法特异性和敏感性好,检测成本低、效率高,适用于禽类常见3种临床重要病原菌单一或混合感染的快速诊断及流行病学调查。

Riemerella anatipestifer, Escherichia coli and Salmonella enteric are three common gram-negative pathogens in birds. In order to detect them rapidly, we developed and optimized a multiple PCR system by detecting their conserved genes, ompA, phoA and fimW, respectively. The results showed that the optimal combination of three pairs of primer was 0.60 μmol/L for R. anatipestifer, 0.28 μmol/L for E. coli, and 0.20 μmol/L for S. enterica, and the optimal annealing temperature was 57℃. The multiple PCR system could differentiate these three specific pathogens from other seven clinical pathogens. The lowest detection limits of the multiple PCR detection system were 1 pg/μL of DNA, and 10^(4)cfu/mL of bacteria. This method showed high specificity, high sensitivity, low costs and high efficiency, and it would be suitable for rapid diagnosis and epidemiological investigation of single or mixed infections of the three important clinical pathogens of birds.