恙虫病东方体超快速检测方法的建立及评价  

Establishment and Evaluation of an Ultra-rapid Method for the Detection of Orientia tsutsugamushi

作　　者：金捷 陆洪斌 张怡[3] 王新宇[3] 吴晶[3] 李涛[3] 姜宁 JIN Jie;LU Hongbin;ZHANG Yi;WANG Xinyu;WU Jing;LI Tao;JIANG Ning(School of Life Sciences,Fudan University,Shanghai 200438,China;The People’s Hospital of Jinping County in Honghe Prefecture of Yunnan Province,Honghe 661599,China;Huashan Hospital Affiliated to Fudan University,Shanghai 200040,China)

机构地区：[1]复旦大学生命科学学院,上海200438 [2]云南省红河州金平县人民医院,红河661599 [3]复旦大学附属华山医院,上海200040

出　　处：《中国生物工程杂志》2024年第4期43-53,共11页China Biotechnology

摘　　要：目的:建立一种可以在基层医疗机构使用的超快速恙虫病东方体(Orientia tsutsugamushi,Ot)核酸检测方法。方法:利用实时荧光定量PCR技术(quantitative real-time polymerase chain reaction,qPCR),通过比对分析数据库中恙虫病东方体全基因组序列确定靶基因,设计qPCR特异性引物和探针,优化反应体系和反应程序,建立超快速多重qPCR检测方法;利用经过数字PCR(droplet digital PCR,ddPCR)定量后的样本核酸作为标准物质,评价该方法的灵敏度、重复性以及最低检出限;通过比较基因组学分析,验证恙虫病东方体与同科属的易引起类似症状的病原体的特异性,通过检测24种(共45例)易引起类似症状的其他病原体核酸评价特异性;利用116例来源于云南省红河州金平县人民医院和复旦大学附属华山医院的临床全血样本,通过四格表卡方检验对多重qPCR超快速检测方法和传统qPCR检测方法的检测结果进行比较,采用Kappa一致性检验分析方法间的一致性。结果:恙虫病东方体与同科属的易引起类似症状的病原体的同源性较低,与常见的易引起类似症状的24种病原体无交叉反应,具有很好的特异性;最低检出限为156 copies/mL;具有很好的重复性,检测Ct值的变异系数≤3.13%;多重qPCR超快速检测方法和传统qPCR检测方法同时检测116例临床全血样本,检测结果的差异无统计学意义(P>0.05),两种方法具有很高的一致性(Kappa=0.9392,P<0.001),总符合率97.42%(CI:92.67%~99.12%),检测时间可缩短至30 min。结论:建立的多重qPCR超快速检测方法具有良好的灵敏度、重复性和特异性,可以快速诊断临床全血样本中的恙虫病东方体核酸,在基层医疗机构实现及时快速诊疗。Objective:To establish an ultra-fast nucleic acid test for Orientia tsutsugamushi that can be used in grassroots medical institutions.Methods:Using quantitative real-time PCR(qPCR),we compared and analyzed the whole genome sequences of Orientia tsutsugamushi in the database,identified the target genes,designed qPCR-specific primers and probes,optimized the reaction system and reaction procedure,and established a multiplex qPCR ultra-rapid assay.The sensitivity,reproducibility,and minimum detection limit of the method were evaluated using viral nucleic acids quantified by droplet digital PCR(ddPCR)as standards.Comparative genomic analysis was performed to verify the specificity of Orientia tsutsugamushi with pathogens of the same family or genus that tend to cause similar symptoms.Specificity was evaluated using nucleic acids from 24(45 cases in total)other pathogens that tend to cause similar symptoms.The results of the multiplex qPCR ultra-rapid assay and the conventional qPCR assay were compared using 116 clinical whole blood samples by four-grid table chi-square test,and the agreement between methods was analyzed using the Kappa consistency test.Results:Orientia tsutsugamushi has low homology to pathogens of the same family or genus that tend to cause similar symptoms.Multiplex qPCR ultra-rapid assay showed good specificity and no cross-reactivity with 24 common pathogens.The minimum detection limit was 156 copies/mL.Repeatability comparison analysis showed a coefficient of variation of≤3.13%for Ct values for each concentration assay.The differences between the multiplex qPCR ultra-rapid assay and the conventional qPCR assay for simultaneous testing of 116 clinical whole blood samples were not statistically significant(P>0.05),the two methods had high agreement(Kappa=0.9392,P<0.001),with total compliance rate 97.42%(CI:92.67%~99.12%),and the testing time could be be reduced to 30 min.Conclusion:The multiplex qPCR ultra-rapid assay has good sensitivity,reproducibility and specificity,which can rapidly diagnos

关 键 词：恙虫病 恙虫病东方体 多重qPCR技术 超快速核酸检测 

分 类 号：Q81[生物学—生物工程]