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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。
In order to establish a double fluorescence quantitative PCR method to detect enterotoxigenic cpe-positive Clostridium perfringens, by comparing some sequences of Clostridium perfringens published in NCBI, the alpha toxin and enterotoxin cpe Based on the conserved sequences, two pairs of primers and one pair of probes were designed and synthesized. The constructed cloning vectors containing α toxin and cpe sequences were used as standards to perform series of optimization and specificity, sensitivity and repeatability tests. The results showed that the method has good specificity and repeatability. The sensitivity of exotoxin cpa was 1.5 × 10 -2 ng / m L and the sensitivity was 1.5 × 10 4 ~ 1.5 × 10 -2 ng / The linear relationship between the Ct value in m L and the logarithm of positive plasmid concentration was 0.991 for R2 value and 1.8 × 10-2 ng / m L for cpe toxin, and the sensitivity was 1.8 × 103-1.8 The Ct value in × 10 ~ (-2) ng / m L showed a good linear relationship with the logarithm of the positive plasmid concentration, and the R2 value reached 0.996. The method was sensitive to bacterial nuclei such as Escherichia coli, Salmonella and Pasteurella Cross-reaction; inter-batch and intra-batch repeatability is good; at the same time, the clinical isolates suspected of clinical isolates were detected by fluorescence quantitative method, the clinical isolates were Clostridium perfringens type A. This double fluorescence quantitative PCR method is sensitive, specific and reproducible.