基于PCR-HRM技术建立石蜡包埋组织低质量样本TP53基因热点变异的检测方法

Enhanced detection of TP53 hotspot mutations in degraded DNA samples from formalin-fixed paraffin-embedded tissues via optimized PCR-HRM protocol

摘要

目的 评价不同组织来源石蜡包埋组织提取的样本 DNA 质量，探讨针对低质量样本 TP53 基因热点变异检测的方法。方法 选择已明确 TP53 热点突变信息的石蜡组织来源 DNA 样本 17 例及 2 例外周血 DNA 样本（对照），均经 Sanger 测序明确突变结果，且这些 DNA 样本均存放于 4 ℃ 环境超过 6 个月，对其进行样本质量评价和 4 个外显子热点突变的再次检测。全部 DNA 均使用同一 DNA 提取试剂盒提取，核酸提取时，石蜡包埋组织的保存时间均超过 5 年。应用核酸定量分析仪和毛细管电泳方法评价 DNA 质量。优化引物、PCR 反应条件、高分辨熔解曲线（HRM）分析等条件，建立低质量样本 TP53 基因热点变异检测方法。结果 石蜡组织 DNA 浓度、纯度虽可达到一般 PCR 扩增要求，但完整性下降，存在不同程度碎片化。与完整性好的 DNA 样本不同，DNA 碎片化程度高时，倍比稀释模板量不能获得理想的扩增标准曲线，甚至高浓度模板可抑制 PCR 反应。通过设计短目标片段（80～140 bp），结合 DNA 质量，优化模板量（降低至 5～10 ng），设立模板倍比稀释的阳性对照和阴性对照辅助结果判定，基于 PCR-HRM 技术，成功实现了碎片化程度不同的 17 例石蜡组织 DNA 样本 TP53 基因热点变异检测。与 Sanger 测序结果比较，17 个样本 4 个外显子，6 个片段检出准确率为 100%（102/102）。结论 针对低质量石蜡组织DNA 样本特点设计，PCR-HRM 技术能够实现临床TP53 变异检测，该技术具有较好的临床推广应用潜能。

Abstract

Objective To evaluate the DNA quality extracted from formalin-fixed paraffin-embedded (FFPE) tissues and explore a method for detecting TP53 hotspot mutations in low quality DNA samples. Methods DNA samples extracted from FFPE tissues of different origins were kept in 4 ℃ fridge for more than 6 months and used in this study. All 19 DNA samples were extracted using the same DNA extracting kit. DNA concentration and purity were determined with a NanoDrop 2000C spectrophotometer. DNA integrity was assessed by capillary electrophoresis using a Bioptic Qseq1 analyzer. TP53 exon 4-7 hotspot mutations were analyzed using Sanger sequencing on all samples. Primers, PCR amplification, and high-resolution melting curve (HRM) analytical conditions were optimized to detect TP53 hotspot mutations in the samples and to establish an enhanced detection method for low quality FFPE samples. Results Although DNA concentration and purity were comparable between FFPE and blood samples, FFPE-derived DNA exhibited varying degrees of fragmentation. Unlike the intact genomic DNA from blood, high DNA fragmentation resulted in reduced PCR amplification efficiency; in some cases, increased template amounts even inhibited the PCR. PCR with a serial dilution template couldn't obtain normal standard amplification curve. By designing short target amplicons (80-140 bp), optimizing the template quantity (5-10 ng) according to DNA quality, and incorporating serially diluted positive and negative controls in HRM analysis, a reliable TP53 hotspot mutation detection method was successfully established. TP53 hotspot mutations were successfully detected in all 17 FFPE DNA samples with different degrees of fragmentation, achieving a 100% accuracy compared to Sanger sequencing results. Conclusion When the experimental design is optimized according to the characteristics of low-quality DNA samples from FFPE tissues, PCR-HRM can be used for reliable detection of TP53 mutations and has good potential for routine clinical molecular diagnosis.

关键词

石蜡包埋组织 / 高分辨熔解曲线 / PCR / TP53

Key words

FFPE / HRM / PCR / TP53

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基金资助

国家自然科学基金面上项目(81972022)

“1+X”大连医科大学附属第二医院自由探索育英项目(LYYH2024006)