羊源多杀性巴氏杆菌重组酶聚合酶扩增诊断方法的建立

Establishment of a Rapid Diagnostic Method of RPA for Pasteurella multocida from Goat

为建立适用于羊源多杀性巴氏杆菌的重组酶聚合酶扩增(RPA)诊断方法,本研究依据前期测序鉴定的羊源多杀性巴氏杆菌KMT1基因序列构建pET-28a(+)-KMT1质粒标准品。设计基于RPA技术的特异性引物及RPA荧光探针,建立实时荧光RPA方法的最适反应体系。采用10倍梯度连续稀释的质粒标准品检测该RPA方法的敏感性并绘制相关性曲线;以10种不同菌株的基因组DNA为模板验证方法的特异性;用感染多杀性巴氏杆菌的山羊及小鼠组织样品对方法的可靠性进行验证。结果显示,本试验建立的实时荧光RPA方法最适反应温度为39℃,最佳引物为KMT1-Fe1,灵敏度达100拷贝/μL,检测下限为10拷贝/μL。与大肠杆菌、金黄色葡萄球菌、副伤寒沙门氏菌、副溶血弧菌、绿脓杆菌、产酸克雷伯菌、布鲁氏菌S2株和鲍曼不动杆菌均无交叉反应。对13个组织样本进行检测,阳性率为76.9%,与实时荧光定量PCR检测结果的符合率达92.3%。综上所述,本研究建立的羊源多杀性巴氏杆菌实时荧光RPA方法具有特异性强、灵敏度高、可靠、快速便捷等特点,适用于多杀性巴氏杆菌的临床分子诊断。

In order to establish a recombinase polymerase amplification(RPA)diagnostic method for Pasteurella multocida from goat,the pET-28a(+)-KMT1 plasmid standard was constructed based on the KMT1 gene sequence of Pasteurella multocida from goat.The specific primers and RPA fluorescent probe based on RPA technology were designed to establish the optimal reaction system of Real-time RPA.The sensitivity of the method was detected and the correlation curve was drawn by 10 fold gradient diluted plasmid standard.The genomic DNA of 10 different strains were used as templates to verify the specificity of the method.Finally,the reliability of the method was verified by the tissue samples of goats and mice infected with Pasteurella multocida.The results showed that the optimal reaction temperature was 39℃,the optimal primer was KMT1-Fe1,and the sensitivity was 100 copies/μL.The detection limit was 10 copies/μL.There was no cross reaction with Escherichia coli,Staphylococcus aureus,Salmonella Paratyphi,Vibrio parahaemolyticus,Pseudomonas aeruginosa,Klebsiella acidogenesis,Brucella S2 and Acinetobacter baumannii.The positive rate of 13 tissue samples was 76.9%,and 92.3%of them were consistent with Real-time PCR.In conclusion,the Real-time RPA of Pasteurella multocida from goat had the characteristics of strong specificity,high sensitivity,good reliability,fast and convenient,which was suitable for clinical molecular diagnosis of Pasteurella multocida.