产气荚膜梭菌肠毒素cpe阳性双重荧光定量PCR方法的建立及应用

发布时间：2018-06-01 19:52

本文选题：产气荚膜梭菌 + 外毒素cpa　；参考：《黑龙江畜牧兽医》2017年01期

【摘要】：为了建立一种双重荧光定量PCR方法检测肠毒素cpe阳性的产气荚膜梭菌,试验通过对部分发表在NCBI中的产气荚膜梭菌序列进行比对,找到其α毒素和肠毒素cpe相对保守的序列,根据保守序列设计合成2对引物和1对探针,以构建的含有α毒素和cpe序列的克隆载体作为标准品,进行系列条件优化及特异性、灵敏性和重复性试验。结果表明:该方法具有很好的特异性和重复性,外毒素cpa的灵敏度为1.5×10~(-2)ng/m L,且在1.5×104~1.5×10~(-2)ng/m L内Ct值与阳性质粒浓度的对数值呈很好的线性关系,R2值达到0.991;肠毒素cpe的灵敏度为1.8×10~(-2)ng/m L,且在1.8×103~1.8×10~(-2)ng/m L内Ct值与阳性质粒浓度的对数值呈很好的线性关系,R2值达到0.996;该方法对大肠杆菌、沙门氏菌、巴氏杆菌等细菌核苷酸无交叉反应;批间和批内重复性良好;同时对实验室保存的临床分离的疑似菌株用建立的荧光定量方法进行检测,结果临床分离菌株均为A型产气荚膜梭菌。说明该双重荧光定量PCR方法灵敏度高、特异性强和重复性好。
[Abstract]:In order to establish a double fluorescence quantitative PCR method for the detection of Clostridium perfringens positive for enterotoxin cpe, the sequence of Clostridium perfringens published in NCBI was compared and the relative conservative sequences of 伪 toxin and cpe were found. Two pairs of primers and one pair of probes were designed and synthesized according to the conserved sequence. The cloned vector containing 伪 toxin and cpe sequence was used as the standard, and a series of optimization conditions, specificity, sensitivity and repeatability tests were carried out. The results show that the method has good specificity and reproducibility. The sensitivity of exotoxin cpa is 1.5 脳 10~(-2)ng/m L, and the logarithmic relationship between Ct value and positive plasmid concentration in 1.5 脳 10 4 10~(-2)ng/m L is very good. R2 is 0.991.The sensitivity of enterotoxin cpe is 1.8 脳 10~(-2)ng/m L, and the logarithm of Ct value and positive plasmid concentration in 1.8 脳 10 3 脳 10~(-2)ng/m L is obtained. The value of R2 was 0.996. The method was applied to the treatment of Escherichia coli. Salmonella, Pasteurella and other bacteria have no cross-reaction with nucleotides, good reproducibility between batches and within batches, and detection of suspected strains of clinical isolates kept in laboratory with established fluorescence quantitative method. Results the clinical isolates were all type A Clostridium perfringens. The results showed that the double fluorescence quantitative PCR method was sensitive, specific and reproducible.
【作者单位】：湖北省农业科学院畜牧兽医研究所/动物胚胎工程及分子育种湖北省重点实验室;
【基金】：公益性行业(农业)科研专项(201303044) 现代农业产业技术体系建设专项(CARS-42-G11) 湖北省农业科学院青年科学基金项目(2014NKYJJ15) 动物胚胎工程及分子育种湖北省重点实验室项目(2016ZD108)
【分类号】：S852.61

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