产气荚膜梭菌多重PCR检测方法的建立及初步应用

Establishment and preliminary application of multiplex PCR for detection of Clostridium perfringens

旨在建立一种高效、快速、准确的检测产气荚膜梭菌并分型的多重PCR方法。根据GenBank上公布的产气荚膜梭菌α、β、ε、ι毒素的基因序列,设计、合成4对针对4种毒素的特异性引物。并通过改变引物浓度、退火温度等反应条件,对多重PCR方法进行优化。结果显示,对产气荚膜梭菌α、β、ε、ι毒素基因均扩增出了预期大小的目的条带,而金黄色葡萄球菌、多杀性巴氏杆菌、停乳链球菌、肺炎克雷伯菌、大肠杆菌均未出现条带;当核酸质量浓度降至12.5μg/L时,仍可清晰地扩增出α、β、ε、ι毒素基因对应条带。在核酸质量浓度降至3.12μg/L时,仍可观察到α与ε毒素基因扩增出的条带。在核酸质量浓度降至0.78μg/L时,α毒素基因对应的条带仍可隐约可见。应用该方法对68份疑似产气荚膜梭菌导致猝死的牛鼻拭子样进行检测,其中有52份样品检测结果显示为C型产气荚膜梭菌,剩余16份样品均为非产气荚膜梭菌,阳性率为76%。结果表明,本研究建立的多重PCR方法特异性强、灵敏度高重复性好,可准确地鉴别区分5种类型的产气荚膜梭菌。在采样时仅需采取疑似病畜的鼻拭子,操作简便、快捷、安全。对临床检测病畜的产气荚膜梭菌病有较为重要的意义。

This study aims to establish an efficient,rapid and accurate multiplex PCR method for detection and typing of Clostridium perfringens.According to the gene sequences ofα,β,εand permethrin of Clostridium perfringens published in GenBank,four pairs of specific primers for the four toxins were designed and synthesized.The multiplex PCR method was optimized by changing the reaction conditions such as primer concentration and annealing temperature.The results showed that the expected bands ofα,β,εand permethrin genes were amplified,but no bands were found in Staphylococcus aureus,Pasteurella multocida,Streptococcus,Klebsiella pneumoniae and Escherichia coli,and the corresponding bands ofα,β,εand permethrin genes could still be amplified clearly when the concentration of nucleic acid decreased to 12.5μg/L.When the concentration of nucleic acid decreased to 3.12μg/L,the bands amplified byαandεtoxin genes could still be observed.When the concentration of nucleic acid decreased to 0.78μg/L,the corresponding band of alpha toxin gene was still vaguely visible.The nasal swab samples of 68 sudden death cattle suspected of Clostridium perfringens infection were detected by this method,of which 52samples showed type C Clostridium perfringens,the remaining 16samples were non-Clostridium perfringens,the positive rate was 76%.The multiplex PCR method established in this study has strong specificity,high sensitivity and good reproducibility,and can accurately identify five types of Clostridium perfringens.Only nose swabs of suspected diseased animals need to be taken when sampling,which is simple,fast and safe.It is of great significance for clinical detection of Clostridium perfringens in diseased animals.