摘要
为建立一种基于铜绿假单胞菌flgE基因的PCR检测方法,本试验根据GenBank中已发表的铜绿假单胞菌flgE基因序列,设计合成了1对特异性引物,由铜绿假单胞菌基因组DNA中扩增获得了目的基因。从退火温度、循环次数、Mg2+浓度、dNTPs浓度和引物浓度5个方面优化了反应条件,并检测了该方法的特异性和敏感性。结果成功扩增出了铜绿假单胞菌1 400 bp的flgE特异性基因片段,最佳退火温度、循环次数、Mg2+浓度、dNTPs浓度和引物浓度分别为56℃、30个循环、1.6 mmol/L、0.2 mmol/L和0.3μmol/L。特异性试验表明,仅铜绿假单胞菌中可扩增出目的片段,而多杀性巴氏杆菌、鼠伤寒沙门氏菌、志贺氏菌、致病性大肠杆菌和金黄色葡萄球菌中均未扩增出相应片段。敏感性试验结果表明,本方法可检测到最低10 pg/μL的铜绿假单胞菌基因组DNA以及100 CFU/mL的病原菌。
To establish a PCR detection method for Pseudomonas aeruginosa(P. aeruginosa), the flgE gene was amplified by PCR using the specific primers which designed according to the sequence logined in GenBank. Then the reaction conditions of PCR were optimized from annealing temperature, cycle times, Mg2+ concentration, dNTPs concentration and primer concentration. Additional, the specificity and sensitivity of PCR were detected. The results showed that the 1 400 bp DNA fragment was specifically amplified from P. aeruginosa. The optimal annealing temperature,cycle times, Mg2+ concentration, dNTPs concentration and primer concentration were 56 ℃, 30 cycles, 1.6 mmol/L, 0.2 mmol/L and 0.3 μmol/L. The result of specificity test showed that target fragment was only amplified from P. aeruginosa and no DNA fragment was obtained from other pathogenic bacteria including Pasteurella multocida, Salmonella pullorum, Shigella, Pathogenic Escherichia coli and Staphylococcus aureus. The sensitivity test showed that the sensitivity of this PCR method was 10 pg/μL and 100 CFU/mL.
作者
宫强
杜珍奇
冯礼尚
樊泽军
冯洋洋
孙鹏飞
程梦琦
Gong Qiang;Du Zhenqi;Feng Lishang;Fan Zejun;Feng Yangyang;Sun Pengfei;Cheng Mengqi(Henan university of Science ang Technology/Henan Engineering Research Center of Food Microbiology,Henan Luoyang 471023)
出处
《现代畜牧兽医》
2020年第3期1-6,共6页
Modern Journal of Animal Husbandry and Veterinary Medicine
基金
河南科技大学SRTP项目(2019178)。
