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

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。

Establishment and Application of PCR Method for Toxin Gene Typing in Clostridium perfringens

This study established a rapid diagnostic PCR method for Clostridium perfringens,provided an effective technical means for the rapid diagnosis and epidemiological investigation of animal Clostridium perfringens infection in order to overcome the long time and high cost disadvantages in traditional identification methods,improve the detection efficiency.Based on the analysis of alpha toxin,beta toxin,epsilon toxin and iota toxin gene sequences for Clostridium perfringens,five pairs of specific primers were designed and synthesized by Premier5.0 software,the PCR method of differential diagnosis for Clostridium perfringens types was established.The optimum annealing temperature was 53 ℃ by repeated experiments.The sensitivity test showed that the PCR can detect the lowest concentrations of DNA of the alpha toxin for 308 pg/L,beta toxin,epsilon toxin for 30.8 pg/L,iota toxin A for 0.122 pg/L,iota toxin B for 0.05 pg/L.The specificity test showed that the method has higher specificity.At the same time,through 16 S rRNA sequence analysis of the positive samples,and compared with GenBank in other Clostridium perfringens 16 S rRNA sequence,the homologies were above 98%.Thus,the established detection method has high sensitivity,strong specificity,and can be used in laboratory diagnosis of Clostridium perfringens infections in animals.