Establishment and performance evaluation of a droplet digital PCR method for detecting of Septin9 gene methylation in colorectal cancer
10.3969/j.issn.1673-4130.2025.06.002
- VernacularTitle: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
- Author:
Jing ZHANG
1
;
Xuyao JI
;
Wenxu YANG
;
Yu LIU
Author Information
1. 哈尔滨医科大学附属第四医院检验科,黑龙江 哈尔滨 150001
- Keywords:
droplet digital PCR;
colorectal cancer;
Septin9 gene methylation;
performance evaluation
- From:
International Journal of Laboratory Medicine
2025;46(6):646-650
- CountryChina
- Language:Chinese
-
Abstract:
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 methyl-ation 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,sensitivi-ty,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 5-104 copy/re-action,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 1.340 0%and 3.330 0%,which met the relevant requirements.The results of accuracy test showed that the positive co-incidence 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 0.881 3(95%CI 0.784-0.978 6).Conclusion The ddPCR method for detection of Septin9 gene methylation in CRC has high sensitivity,speci-ficity,precision,accuracy and clinical diagnostic accuracy.It can provide a reliable technical means for early di-agnosis,monitoring and treatment of CRC.
