一种新型基于微流控芯片的数字 PCR 方法，通过 mSEPT9 提高结直肠癌早期诊断的灵敏度。
A novel microfluidic chip-based digital PCR method for enhanced sensitivity in the early diagnosis of colorectal cancer via mSEPT9.
机构信息
Department of Laboratory Medicine, Fujian Key Laboratory of Laboratory Medicine, Gene Diagnosis Research Center, Fujian Clinical Research Center for Laboratory Medicine of Immunology, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Fujian, China; Department of Laboratory Medicine, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou 350212, Fujian, China.
Department of Laboratory Medicine, Fujian Key Laboratory of Laboratory Medicine, Gene Diagnosis Research Center, Fujian Clinical Research Center for Laboratory Medicine of Immunology, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Fujian, China; Department of Laboratory Medicine, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou 350212, Fujian, China; The First Clinical College, Fujian Medical University, Fuzhou 350005, Fujian, China.
出版信息
Clin Chim Acta. 2024 Feb 1;554:117781. doi: 10.1016/j.cca.2024.117781. Epub 2024 Jan 14.
BACKGROUND
To enhance the sensitivity of plasma methylated Septin9 gene (mSEPT9) detection in colorectal cancer (CRC) screening, we developed a microfluidic chip-based digital PCR (dPCR) method suitable for low-concentration samples, aiming to apply it for mSEPT9 detection in CRC diagnosis.
METHODS
Our microfluidic chip-based dPCR method utilized specific primers and probes with locked nucleic acids (LNAs) modifications for mSEPT9 detection. We evaluated its performance, including detection limit, specificity, and linear range, comparing it with a commercial qPCR reagent kit using the same samples (95 CRC, 23 non-CRC).
RESULTS
The LNAs-modified dPCR method showed a linear range of 10-10 copies/μL and a detection limit of 10 copies/μL. Clinical testing revealed that our dPCR method exhibited a sensitivity of 82.11 % and specificity of 95.65 % for CRC diagnosis, outperforming the commercial qPCR kit (sensitivity: 58.95 %, specificity: 91.30 %), particularly in Stage I with a diagnostic sensitivity of 90.91 %. Combining mSEPT9 and carcinoembryonic antigen (CEA) improved diagnostic sensitivity to 91.49 %.
CONCLUSIONS
Our accurate microfluidic chip-based dPCR method, especially in combination with CEA, holds promise for effective CRC screening and timely interventions, offering enhanced mSEPT9 quantification over conventional qPCR.
背景
为了提高血浆中甲基化 Septin9 基因（mSEPT9）检测在结直肠癌（CRC）筛查中的灵敏度，我们开发了一种适用于低浓度样本的基于微流控芯片的数字 PCR（dPCR）方法，旨在将其应用于 CRC 诊断中的 mSEPT9 检测。
方法
我们基于微流控芯片的 dPCR 方法利用带有锁核酸（LNA）修饰的特异性引物和探针来检测 mSEPT9。我们评估了其性能，包括检测限、特异性和线性范围，并使用相同的样本（95 例 CRC、23 例非 CRC）与商业 qPCR 试剂试剂盒进行比较。
结果
LNA 修饰的 dPCR 方法显示线性范围为 10-10 拷贝/μL，检测限为 10 拷贝/μL。临床检测表明，我们的 dPCR 方法对 CRC 诊断的灵敏度为 82.11%，特异性为 95.65%，优于商业 qPCR 试剂盒（灵敏度：58.95%，特异性：91.30%），特别是在 I 期诊断灵敏度为 90.91%。结合 mSEPT9 和癌胚抗原（CEA）可将诊断灵敏度提高至 91.49%。
结论
我们准确的基于微流控芯片的 dPCR 方法，特别是与 CEA 联合使用，有望实现有效的 CRC 筛查和及时干预，与传统 qPCR 相比，可更准确地定量 mSEPT9。
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