产气荚膜梭菌毒素基因分型PCR检测方法的建立及初步应用

发布时间：2018-03-25 02:25

  本文选题：产气荚膜梭菌　切入点：毒素基因　出处：《动物医学进展》2017年03期

【摘要】：建立一种快速鉴别诊断不同型产气荚膜梭菌的PCR检测方法,为动物产气荚膜梭菌病的快速诊断及流行病学调查提供有效的技术手段。克服传统鉴定方法耗时长、费用高的缺点,提高了检测效率。通过对产气荚膜梭菌α毒素、β毒素、ε毒素和ι毒素基因序列分析,利用Premier5.0软件设计并合成了5对特异性引物,建立了针对5种不同型产气荚膜梭菌的PCR鉴别诊断方法。通过反复试验确定了最佳退火温度为53℃。通过灵敏度试验表明,PCR检测方法最低能检测到的DNA浓度α毒素为308pg/μL,β毒素、ε毒素为30.8pg/μL,ι毒素A为0.122pg/μL,ι毒素B为0.05pg/μL。通过特异性试验表明,本方法具有较高的特异性。同时,通过对本方法检测出的阳性样品16S rRNA序列分析发现,与GenBank中的其他产气荚膜梭菌的16S rRNA序列同源性均在98%以上。表明建立的检测方法灵敏度高、特异性强,可以应用于动物产气荚膜梭菌病的实验室诊断。
[Abstract]:A rapid PCR detection method for the differential diagnosis of different Clostridium perfringens was established to provide an effective technique for the rapid diagnosis and epidemiological investigation of Clostridium perfringens in animals, and to overcome the disadvantages of long time and high cost in traditional identification methods. The detection efficiency was improved. By sequence analysis of 伪 toxin, 尾 toxin, 蔚 toxin and l toxin genes of Clostridium perfringens, 5 pairs of specific primers were designed and synthesized by Premier5.0 software. A method for the differential diagnosis of 5 different Clostridium perfringens by PCR was established. The optimum annealing temperature was determined to be 53 鈩，

本文编号：1661134