微滴式数字PCR技术快速诊断侵袭性念珠菌病的方法建立  

ARapid diagnosis of invasive candidiasis by droplet digital PCR

作　　者：何志捷 李伟超[2] 何铭辉 陈晓彤[2] 林钊 植耀炜[2] HE Zhijie;LI Weichao;HE Minghui;CHEN Xiaotong;LIN Zhao;ZHI Yaowei(Faculty of Rehabilitaion Medicine,Guangzhou Xinhua University,Guangzhou 510520,Guangdong,China;不详)

机构地区：[1]广州新华学院康复医学系,广东广州510520 [2]中山大学孙逸仙纪念医院,广东广州510000 [3]广州永诺医学检验所有限公司,广东广州510000

出　　处：《实用医学杂志》2024年第19期2738-2746,共9页The Journal of Practical Medicine

基　　金：广东省基础与应用基础研究基金项目(编号:2022A1515011248)。

摘　　要：目的建立基于微滴式数字PCR(droplet digital PCR,ddPCR)技术快速检测重症患者侵袭性念珠菌感染的方法。方法基于微滴式数字PCR平台建立检测体系,设计四种念珠菌(白色念珠菌、光滑念珠菌、近平滑念珠菌和热带滑念珠菌)特异性引物探针;(1)对无模板对照(no tmplate contral,NTC)样品进行检测,确定空白限(limit of blank,LOB)范围和阳性判断值;(2)通过对阳性样本进行稀释,每个浓度梯度各进行10次重复提取检测,确定检测限(limit of detection,LOD)范围;(3)重复检测稀释后的样本,确定定量限(limit of quantitation,LOQ)范围;(4)对阳性样品进行梯度稀释,确定线性范围;(5)通过2个高低浓度的阳性样本提取检测,进行12次重复性测试,计算结果浓度对数值的变异系数(CV);(6)通过具有真菌培养结果的临床样本进行检测,评估方法可靠度。结果ddPCR检测念珠菌LOB在0~81 copies/mL之间,阳性判断值为≥3个阳性微滴;LOD为3×10^(2) copies/mL;LOQ为3×10^(2) copies/mL;不同浓度梯度检测线性范围是3×10^(2)~3×10^(7)copies/mL,相关系数为:白色念珠菌R^(2)=0.9995、光滑念珠菌R^(2)=0.9989、近平滑念珠菌R^(2)=0.9994、热带滑念珠菌R^(2)=0.999;结果浓度对数值的CV<5%,满足精密度要求;通过检测建立方法初步验证临床标本,结果与临床培养结果一致。结论ddPCR对重症患者侵袭性念珠菌感染的检测灵敏度高、重复性好,特异性高。Objective To establish a rapid detection method for invasive candidiasis based on droplet digital polymerase chain reaction(ddPCR).Methods We developed an assay system using a microtitre-based digital PCR platform and designed primer probes specific for four Candida species,namely Candida albicans,Candida smoothii,Candida near-smoothii,and Candida tropicalis.(1)The Limit of Blank(LOB)range and positive judg-ment value were determined by analyzing No Template Control(NTC)samples.(2)The Limit of Detection(LOD)range was determined by diluting positive samples with 10 replicate extractions at each concentration gradient.(3)The Linear Limit of Quantitation(LOQ)range was determined by repetitive testing of diluted samples.(4)The linear range limit was determined through gradient dilution of the positive samples.(5)The coefficient of variation(CV),calculated from the logarithmic values of the resultant concentrations,was assessed by extracting and test-ing positive samples in 12 repetitions at both high and low concentrations.(6)Method reliability was evaluated by calculating the CV from the logarithmic values of the resultant concentrations obtained from clinical samples with fungal culture results.Results The ddPCR assay detected Candida LOB at a range of 0~81 copies/mL,with a positive threshold set at≥3 positive microdroplets.The LOD and LOQ were determined to be 3×10^(2) copies/mL.The linear range for detecting different concentration gradients was found to be between 3×10^(2) and 3×10^(7) copies/mL,with high correlation coefficients observed for Candida albicans(R^(2)=0.9995),Candida smoothii(R^(2)=0.9989),Candida near-smoothii(R^(2)=0.9994),and Candida tropicalis(R^(2)=0.999).Additionally,the coefficient of variation for the resultant concentration logarithmic values was less than 5%,meeting precision requirements.Furthermore,preliminary validation using clinical specimens demonstrated consistent results compared to clinical culture findings.Conclusion ddPCR exhibits rapidity,high sensitivity,good repeatabili

关 键 词：微滴式数字PCR 侵袭性念珠菌病 快速诊断 

分 类 号：R446[医药卫生—诊断学]