微滴式数字PCR检测结直肠癌Septin9基因甲基化的方法建立及性能评价  

Establishment and performance evaluation of a droplet digital PCR method for detecting of Septin9 gene methylation in colorectal cancer

作　　者：张静[1] 吉旭瑶 杨文旭 刘禹[1] ZHANG Jing;JI Xuyao;YANG Wenxu;LIU Yu(Department of Clinical Laboratory,the Fourth Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150001,China)

机构地区：[1]哈尔滨医科大学附属第四医院检验科,黑龙江哈尔滨150001

出　　处：《国际检验医学杂志》2025年第6期646-650,共5页International Journal of Laboratory Medicine

基　　金：国家自然科学基金项目(82272389)。

摘　　要：目的建立微滴式数字PCR(ddPCR)检测结直肠癌(CRC)的Septin9基因甲基化的方法,并对其性能进行评价。方法针对Septin9基因甲基化设计特异的引物与探针,并优化ddPCR检测Septin9基因甲基化反应条件,包括反应的引物与探针浓度、退火温度与循环数,建立ddPCR检测Septin9基因甲基化检测方法。通过检测甲基化标准物质及临床标本,评价所建方法的线性、灵敏度、特异度、精密度、准确度及临床诊断准确性。结果ddPCR检测Septin9基因甲基化反应的最优引物与探针浓度分别为500 nmol/L与200 nmol/L,最佳退火温度为56℃,最佳循环数为45次。线性试验表明,在5~10^(4)拷贝/反应内,线性表现良好。ddPCR检测Septin9基因甲基化浓度预设检出限为0.422%,且能特异识别Septin9基因甲基化阳性质控品。精密度评价中,高浓度和低浓度参考品的实验室变异系数分别为1.3400%与3.3300%,满足相关要求。准确度试验结果显示,3个批次的甲基化质控样品10次重复检测的阳性符合率和阴性符合率均为100%。临床标本检测结果显示,ddPCR检测Septin9基因甲基化对CRC组的灵敏度为82.61%(95%CI 66.10%~99.10%),特异度为78.38%(95%CI 65.20%~91.60%),曲线下面积为0.8813(95%CI 0.7840~0.9786)。结论该研究建立的ddPCR检测CRC的Septin9基因甲基化的方法具有较高的灵敏度、特异度、精密度、准确度及临床诊断准确性,可为CRC的早期诊断和监测治疗提供可靠的技术手段。Objective To establish a droplet digital PCR(ddPCR)method for detecting the Septin9 gene methylation in colorectal cancer(CRC)and evaluate its performance.Methods Specific primers and probes were designed for the methylation of Septin9 gene.The reaction conditions of ddPCR for Septin9 gene methylation detection were optimized,including primer and probe concentrations,annealing temperature and cycle number.A ddPCR method to detect the methylation of Septin9 gene was established.The linearity,sensitivity,specificity,precision,accuracy and clinical diagnostic accuracy of the established method were evaluated by detecting methylation reference materials and clinical samples.Results The optimal concentrations of primers and probes were 500 nmol/L and 200 nmol/L,respectively.The optimal annealing temperature was 56℃and the optimal number of cycles was 45.The linearity test showed good linearity in the range of 5-10^(4) copy/reaction,and the preset detection limit of Septin9 gene methylation concentration detected by ddPCR was 0.422%,which could specifically identify Septin9 gene methylation positive control.In precision evaluation,the coefficients of variation of high concentration and low concentration reference materials were 1.3400%and 3.3300%,which met the relevant requirements.The results of accuracy test showed that the positive coincidence rate and negative coincidence rate of 10 repeated tests of methylation quality control samples of 3 batches were both 100%.The results of clinical samples showed that the sensitivity and specificity of ddPCR detection of Septin9 gene methylation in CRC group were 82.61%(95%CI 66.10%—99.10%)and 78.38%(95%CI 65.20%—91.60%),respectively.The area under the curve was 0.8813(95%CI 0.784—0.9786).Conclusion The ddPCR method for detection of Septin9 gene methylation in CRC has high sensitivity,specificity,precision,accuracy and clinical diagnostic accuracy.It can provide a reliable technical means for early diagnosis,monitoring and treatment of CRC.

关 键 词：微滴式数字PCR 结直肠癌 Septin9基因甲基化 性能评价 

分 类 号：R446.9[医药卫生—诊断学]