摘要目的 建立一种基于重组酶介导扩增技术(RAA)与分子信标相结合的病原菌恒温快速检测方法.方法 方法学的建立.通过金黄色葡萄球菌A蛋白(SPA)所对应的基因设计相应引物及分子信标探针,不断调整引物浓度比例以确定最佳引物浓度比来进行不对称扩增,利用不对称重组酶介导扩增并与分子信标探针杂交,通过琼脂糖凝胶电泳及荧光采集观测结果.将阳性质粒以10倍的梯度稀释,检测该方法的灵敏度.利用该RAA杂交法检测大坪医院检验科微生物室保存的2016年12月的72株包含金黄色葡萄球菌及葡萄球菌属其他种细菌的菌株,以检测该方法的特异性.在特异性实验基础上添加我室保存的2016年12月的39例其他菌株进行Kappa一致性检验及临床诊断效能评价.结果 当限制性引物与非限制性引物的浓度比例在1:20时,产生单链DNA(ssDNA)的效率最高.不对称RAA扩增与分子信标探针杂交的效率明显高于对称扩增.该方法的灵敏度为20拷贝/μl.RAA杂交法能够将金黄色葡萄球菌与葡萄球菌属其他菌株区分,与传统金标准相比Kappa为0.860,一致性良好.经过对111株菌株的检测,该方法敏感度为92.5％ (37/40),特异度为97.2％(69/71),阳性预测值为94.9％(37/39),阴性预测值为95.8％ (69/72),阳性似然比为33.04,阴性似然比为0.077,约登指数为0.897.与传统金标准相比,Kappa为0.902,两种方法一致性良好.结论 通过对不对称RAA扩增条件的优化及与分子信标探针的结合,建立了RAA杂交技术检测细菌DNA的新方法,为恒温扩增应用于临床奠定基础.

abstractsObjective To establish a homothermal and fast detecting method on pathogenic bacteria by combining recombinase-aid amplification (RAA) with molecular beacon.Methods The establishment of the methodology.Staphylococcus aureus specific primers were designed from the relative region of the staphylococcal protein A (SPA).Asymmetry amplification was optimized by adjusting the primer concentration ratios.The results of amplification and hybridization were visualized and analyzed by agarose gel electrophoresis and fluorescence detection.The sensitivity was identified by detecting dilute positive plasmids.And the specificity was determined using RAA method by detecting 72 pathogenic bacteria,including Staphylococcus aureus and other Staphylococcus spp.from the Department of Clinical Laboratory of Daping Hospital in December 2016.Besides,the Kappa analysis and the clinical diagnosis efficiency were investigated by analyzing 39 extra strains in the laboratory in December 2016.Results When the concentration ratio of restrictive and non-restrictive primer was 1:20,the yield efficiency of single-stranded DNA (ssDNA) reached the peak.And as for the hybridization efficiency,the asymmetry amplification was higher than symmetry amplification.Twenty copies/μl was proposed as the limits of detection by testing dilute plasmids.And the RAA hybridization method could distinguish Staphylococcus aureus with other Staphylococcus spp.Comparing with traditional detection methods with a Kappa index of 0.860,this method shows a good consistency.By analyzing the 111 bacteria,the sensitivity of the method is 92.5％ (37/40),the specificity is 97.2％ (69/71),the positive predictive value is 94.9％ (37/39),the negative predictive value is 95.8％ (69/72),the positive likelihood radio is 33.04,the negative likelihood radio is 0.077,the Youden index is 0.897 and the Kappa index is 0.902.Conclusion Through the combination of asymmetry recombinase-aid amplification optimization and molecular beacon probe,a new method of detecting bacteria DNA with RAA hybridization technique is established,providing the foundation for its clinical application.