SYBR Green qPCR 检测内镜活检 胃组织石蜡标本幽门螺杆菌

SYBR Green qPCR Detection of Helicobacter Pylori in Paraffin Sections of Endoscopic Biopsy Gastric Tissue

摘要:

〔摘 要〕 目的：建立一种高效、特异的检测内镜活检胃组织石蜡包埋标本幽门螺杆菌（Hp）实时荧光定量聚合酶链 式反应（qPCR）方法。 方法：针对 Hp 23S 核糖体核糖核酸（rRNA）基因设计特异度引物，建立 SYBR Green qPCR 反应 体系，并验证该方法的特异度、灵敏度和重复性。用该方法对 20 份内镜活检胃组织石蜡标本进行检测，同时作免疫组织 化学染色分析。阳性扩增产物以 Sanger 测序法明确克拉霉素基因突变情况。 结果：本研究 qPCR 方法具备良好特异度和灵 敏度，最低可检测 2.3×10² copies·μL^-1 的质粒脱氧核糖核酸（DNA）。标准曲线表明其具有良好线性关系，直线方程为 y = –3.6106x + 44.626，R² 为 0.9879 ＞ 0.98。不同浓度标准品的批内变异系数为 0.20 % ~ 2.19 %，批间变异系数为 1.24 % ~ 2.63 %，均＜ 3 %，表明该方法的稳定性和重复性良好。针对 20 份疑似感染 Hp 的内镜活检胃黏膜组织石蜡标本，该方法 检出 Hp 阳性样品 14 份，阳性检出率为 70 %，免疫组织化学染色方法检出 Hp 阳性样品 15 份，阳性检出率为 75 %，二者 的符合率为 93.33 %。阳性扩增产物经 Sanger 测序法测序显示均为 Hp 23S rRNA 基因片段，且 14 例样品中有 5 例存在克拉 霉素耐药基因突变。 结论：本研究建立的 SYBR Green qPCR 方法的特异度、灵敏度和重复性良好，后续阳性扩增产物的测 序结果可明确克拉霉素耐药基因突变情况，适用于临床 Hp 感染和克拉霉素耐药性的检测。

Abstract:

〔Abstract〕 Objective To develop a highly efficient and specific real-time quantitative polymerase chain reaction (qPCR) method for the detection of Helicobacter pylori (Hp) in paraffin-embedded gastric tissue samples obtained through endoscopic biopsy. Methods Specific primers were designed for Hp 23S ribosomal ribonucleic acid (rRNA) gene, and SYBR Green qPCR reaction system was established, and the specificity, sensitivity and repeatability of the method were verified. This method was used to detect paraffin wax in 20 gastric tissues from endoscopic biopsy, and immunohistochemical analysis was performed. The mutation of clarithromycin gene was determined by Sanger sequencing. Results The qPCR method exhibited excellent specificity and sensitivity, with a lowest detection limit of 2.3×10² copies·μL^-1 of plasmid deoxyribonucleic acid (DNA). The standard curve demonstrated good linearity, with the linear equation y = -3.6106x + 44.626 and an R² value of 0.9879, exceeding 0.98. The intrabatch coefficients of variation for different concentrations of standard samples ranged from 0.20% to 2.19%, and the inter-batch coefficients of variation ranged from 1.24% to 2.63%, both less than 3%, indicating excellent stability and repeatability of the method. Among the 20 suspected Hp-infected paraffin-embedded gastric mucosal tissue samples obtained through endoscopic biopsy, the qPCR method detected Hp-positive samples in 14 cases, resulting in a positive detection rate of 70%. The immunohistochemical staining method detected Hp-positive samples in 15 cases, with a positive detection rate of 75%, and the concordance rate between the two methods was 93.33%. Sequencing of positive amplification products using the Sanger method confirmed the presence of Hp 23S rRNA gene segments in all 14 samples, with 5 cases showing clarithromycin-resistant gene mutations. Conclusion The SYBR Green qPCR method established in this study exhibited excellent specificity, sensitivity, and repeatability. Additionally, the subsequent sequencing of positive amplification products provided clarity on the presence of clarithromycin-resistant gene mutations,making it suitable for clinical detection of Hp infection and clarithromycin resistance.