基于多交叉置换扩增和纳米生物传感技术快速检测肺炎支原体方法的建立  

Establishment of rapid detection of mycoplasma pneumoniae on the basis of multiple cross displacement amplification and nanoparticle-based biosensor

作　　者：肖飞[1] 郑宝英[2] 徐文健[3] 伏瑾[1] 黄小兰[1] 孙春荣[1] 贾楠 张裕[1] 许峥[1] 周娟 王毅 Xiao Fei;Zheng Baoying;Xu Wenjian;Fu Jin;Huang Xiaolan;Sun Chunrong;Jia Nan;Zhang Yu;Xu Zheng;Zhou Juan;Wang Yi(Experimental Center of Capital Institute of Pediatrics,Beijing 100020;Department of Respiratory,Children’s Hospital Affiliated to Capital Institute of Pediatrics,Beijing 100020;Testing Center of Children’s Hospital Affiliated to Capital Institute of Pediatrics,Beijing 100020)

机构地区：[1]首都儿科研究所实验中心,北京100020 [2]首都儿科研究所附属儿童医院呼吸科,北京100020 [3]首都儿科研究所附属儿童医院检验中心,北京100020

出　　处：《遵义医科大学学报》2024年第5期513-521,共9页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:82200115)。

摘　　要：目的建立一种简单、灵敏、快速的肺炎支原体(MP)检测方法,并对其应用性进行验证和评价。方法利用多交叉置换扩增(MCDA)技术对肺炎支原体特异基因CARDS毒素基因进行扩增,利用侧流免疫层析生物传感(LFB)技术读取扩增结果,命名该方法为MP-MCDA-LFB。分析扩增反应在60~67℃(间隔1℃)的扩增效率,筛选最适反应温度;分析分别扩增10、20、30、40 min时能够检测到的最低核酸浓度,筛选最佳反应时间。利用10倍系列稀释的肺炎支原体核酸分析MP-MCDA-LFB方法的灵敏度和检测限,利用35株非肺炎支原体菌株分析MP-MCDA-LFB方法的特异性。利用MP-MCDA-LFB方法检测80份疑似MP感染的临床样本,并与RT-PCR法检测结果进行比较,分析MP-MCDA-LFB方法的临床应用性。结果MP-MCDA-LFB能够实现对肺炎支原体CARDS毒素基因的快速检测。其最佳反应温度为63℃,最短反应时间为40 min,整个检测过程可在1 h内。MP-MCDA-LFB方法具有较高的灵敏度和特异性,其检测限低至45 ng/L,与其他临床表现相似的病原体无交叉反应,特异性为100%。MP-MCDA-LFB方法从80份临床样本中检出45份阳性样本(56.3%),检出率与RT-PCR方法一致。结论本研究建立的以CARDS毒素基因为靶标的MP-MCDA-LFB检测方法具有简单、快速、灵敏度高、特异性强的优点,在基层医疗机构和现场检测具有较好的应用潜力。Objective To establish a simple,sensitive and rapid detection method for Mycoplasma pneumoniae(M.pneumoniae)and evaluate its application.Methods Multiple cross-displacement amplification(MCDA)reaction was used to amplify the CARDS toxin gene specific for M.pneumoniae,and lateral flow biosensor(LFB)technique was used to interpret the amplicons,and the method was termed as MP-MCDA-LFB.Assays performed at 60-67℃were analyzed to obtain the optimal reaction temperature,and amplicons within different reaction time(10-40 min,10 min interval)were readout to obtain the optimal reaction time for MP-MCDA-LFB assay.Serial dilutions of genomic DNA of M.pneumoniae were prepared for analytical sensitivity analysis and 35 non-M.pneumoniae strains were selected for analytical specificity analysis.Moreover,the MP-MCDA-LFB method was further utilized to diagnose 80 clinical samples,then the obtained results were compared with that by RT-PCR method to validate the clinical feasibility of the new method.Results The MP-MCDA-LFB method was confirmed feasible for quick diagnosis of M.pneumoniae.The optimum reaction temperature was 63℃,and the optimal reaction time was 40 min,enabling the whole detection completed within 1 h.The MP-MCDA-LFB assay was confirmed highly sensitive and specific for M.pneumoniae detection,with a limit of detection at 45 ng/L and a specificity of 100%,displaying no cross-reaction with non-M.pneumoniae strains.In addition,the MP-MCDA-LFB assay detected 45 positive samples from the 80 clinical samples(56.3%),which was completely in accordance with that of RT-PCR assay.Conclusion The MP-MCDA-LFB detection method targeting CARDS toxin gene established in this study has the characteristics of simple,rapid,highly sensitive and strongly specific,exhibiting preferable potential of application in primary medical institutions and point-of-care tests.

关 键 词：肺炎支原体 多交叉置换扩增技术 侧流免疫层析生物传感技术 CARDS毒素基因 RT-PCR 

分 类 号：R375.2[医药卫生—病原生物学]