基于实时荧光重组酶聚合酶扩增(RPA)技术的金黄色葡萄球菌快速检测方法的建立

Establishment of a rapid detection method for Staphylococcus aureus based on real-time fluorescent recombinase polymerase amplification(RPA)

目的建立一种针对金黄色葡萄球菌(Staphylococcus aureus,SA)辅助耐药基因femB的实时荧光重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术检测方法,并评价其灵敏度、特异性以及实用性。方法针对femB特异基因设计RPA候选扩增引物以及RPA荧光探针,设置同一浓度的阳性质粒浓度筛选出最优的RPA扩增引物,建立SA的实时荧光RPA(exo-RPA)检测方法,并设置SA基因组DNA浓度梯度测试该检测方法的灵敏度;选用11种其他致病菌基因组DNA测试方法特异性。最后模拟临床血液样本比较该法与qPCR检测方法的阳性检出率,评价方法的实用性。结果SA-exo-RPA检测方法在39℃反应温度下,5~10 min内可快速检出最低浓度为1 amol/L(3.82 fg)的SA基因组DNA,且特异性高,与其他致病菌无交叉反应。模拟临床血液样本阳性检出率与已发表文献中qPCR检测方法一致,不受样本中复杂成分的非靶标核酸干扰。结论该研究建立的SA-exo-RPA检测方法具有快速、高灵敏度和高特异性的优点,可应用于SA的现场快速检测。

Objective To establish a real-time recombinase polymerase amplification(RPA)detection method for SA assisted drug resistance gene femB,and evaluate its sensitivity,specificity and practicability.Methods RPA candidate amplification primers and RPA fluorescent probes were designed for femB,and the optimal RPA amplification primers were screened by setting the same concentration of the positive plasmid to establish an SA-exo-RPA detection method.The sensitivity of this detection method was tested by setting the concentration gradient of SA genomic DNA.The genomic DNA mixture of 11 other pathogens was used to test the specificity of this method.Finally,the positive detection rates of this method and qPCR detection method were compared by simulating clinical blood samples,and the practicability of this method was evaluated.Results This SA-exo-RPA detection method could rapidly detect SA genomic DNA at a minimum concentration of 1 amol/L(3.82 fg)within 5-10 min at 39℃,with a high specificity and no cross-reaction with other pathogenic bacteria.The positive detection rate of simulated clinical blood samples was consistent with that of qPCR detection methods in published literature,and was not affected by complex components of samples.Conclusion The SA-exo-RPA detection method established in this study is characterized by a high sensitivity and specificity,rapidity and simplicity,which can be applied to the field rapid detection of SA.