【摘要】 目的 针对肺炎链球菌自溶素lytA基因,建立一种基于重组酶介导等温核酸扩增(RAA)联合基于激烈火球菌Argonaute蛋白(PfAgo)技术的检测方法,并评价该方法的灵敏度、特异度及临床适用性。方法根据肺炎链球菌自溶素lytA基因序列,设计5对RAA特异性引物和3组引导DNA(gDNA),建立并优化RAA-PfAgo技术,最后,用该技术对细菌菌株和临床样本进行检测,以确定该技术的灵敏度和特异度。结果 将RAA技术、PfAgo技术与Chelex-100核酸粗提取法联合,无需在专业实验室中进行检测,检测限为1.5×10~1CFU/mL。该技术能特异性地检测肺炎链球菌。以细菌培养法的检测结果作为参考标准,88份临床样本的符合率为96.59%,灵敏度为100.00%,特异度为93.48%。该技术与聚合酶链反应(PCR)检测结果高度一致,且相较于细菌培养法具有更高的灵敏度。结论 RAA-PfAgo技术检测肺炎链球菌灵敏度和特异度均较高,可应用于肺炎链球菌的快速检测。更多还原

【Abstract】 Objective To establish a detection method based on recombinase aided amplification(RAA)combined with Pyrococcus furiosus Argonaute(PfAgo) technology for the autolosyin lytA gene of Streptococcus pneumoniae,and to evaluate the sensitivity,specificity,and clinical applicability of the method.Methods A total of 5 pairs of RAA specific primers and 3 sets of guide DNA(gDNA) were designed based on the autolosyin lytA gene sequence of Streptococcus pneumoniae to establish and optimize the RAA-PfAgo technology.Finally,the method was used to detect bacterial strains and clinical samples to determine the sensitivity and specificity of the method.Results The RAA technology,PfAgo technology and the Chelex-100crude nucleic acid extraction method were combined.The experiment did not require detection in a professional laboratory.The detection limit was 1.5×10~1 CFU/mL.The method could specifically detect Streptococcus pneumoniae;and the bacterial culture results were used as a reference standard,and the conformity rate of 88clinical samples was 96.59%.The sensitivity was 100.00% and the specificity was 93.48%.The method demonstrated high concordance with PCR results and exhibited greater sensitivity compared to the bacterial culture method.Conclusion The RAA-PfAgo technology exhibits high sensitivity and specificity in detecting Streptococcus pneumoniae,and is expected to be applied in the rapid detection of this bacterium.更多还原