Li Jinqian, Liang Jiaqi, Xu Yinyan, Tan Wei, Chen Guanzheng, Ou Tong
Medical Laboratory, Shenzhen Luohu People's Hospital, Shenzhen, Guangdong, 518000, China.
NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University, Haikou, Hainan, 571199, China.
BMC Cancer. 2025 Jul 24;25(1):1211. doi: 10.1186/s12885-025-14611-0.
To establish and validate a droplet digital PCR (ddPCR) assay for quantifying FRS2 gene copy number in formalin-fixed paraffin-embedded (FFPE) bladder cancer tissue samples, and to evaluate its analytical performance.
The ddPCR assay was developed using FRS2 as the target gene and RPP30 as the reference gene. Artificial plasmids, genomic DNA from urinary sediment of healthy individuals and cell lines were used as templates to assess the assay's precision, minimum reliable input DNA, and linearity. Fluorescence in situ hybridization (FISH) was employed to validate the accuracy of the ddPCR results.
One-dimensional fluorescence amplitude plots showed clear separation between positive and negative droplets for both FRS2 and RPP30. Duplex detection of FRS2 and RPP30 within the same reaction showed no interference between primers or probes. The assay exhibited excellent repeatability and precision, with intra-assay coefficient of variation (CV)% of 2.58% and 3.75%, and inter-assay CV% of 2.68% and 3.79%, across 20 ng and 2 ng input levels, respectively. The minimum reliable input DNA amount was determined to be 2 ng, and a strong linear relationship was observed (R >0.99). Compared to FISH, the ddPCR assay showed 100% sensitivity, 100% specificity, and a kappa value of 1.
The developed ddPCR assay enables accurate and reliable quantification of FRS2 copy number in FFPE samples, offering a promising tool for auxiliary diagnosis and prognostic assessment in bladder cancer.
建立并验证一种用于定量福尔马林固定石蜡包埋（FFPE）膀胱癌组织样本中FRS2基因拷贝数的液滴数字PCR（ddPCR）检测方法，并评估其分析性能。
以FRS2为靶基因、RPP30为参照基因开发ddPCR检测方法。使用人工质粒、健康个体尿沉渣基因组DNA和细胞系作为模板，评估该检测方法的精密度、最低可靠输入DNA量和线性度。采用荧光原位杂交（FISH）验证ddPCR结果的准确性。
一维荧光幅度图显示FRS2和RPP30的阳性和阴性液滴之间有明显分离。在同一反应中对FRS2和RPP30进行双重检测，引物或探针之间无干扰。该检测方法具有出色的重复性和精密度，在20 ng和2 ng输入水平下，批内变异系数（CV）%分别为2.58%和3.75%，批间CV%分别为2.68%和3.79%。确定最低可靠输入DNA量为2 ng，观察到强线性关系（R>0.99）。与FISH相比，ddPCR检测方法显示出100%的灵敏度、100%的特异性和1的kappa值。
所开发的ddPCR检测方法能够准确可靠地定量FFPE样本中的FRS2拷贝数，为膀胱癌的辅助诊断和预后评估提供了一种有前景的工具。
