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

作　　者：郑玮 李倩 王营辉 ZHENG Wei;LI Qian;WANG Yinghui(Zhongshan Hospital Xiamen University,Fujian Xiamen 361000)

机构地区：[1]厦门大学附属中山医院,福建厦门361000

出　　处：《深圳中西医结合杂志》2023年第20期55-58,I0005,共5页Shenzhen Journal of Integrated Traditional Chinese and Western Medicine

基　　金：福建省卫生健康青年科研项目(2022QNB015)。

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2.3×10^(2)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。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 2.3×10^(2)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 clari

关 键 词：幽门螺杆菌 SYBR Green实时荧光定量聚合酶链式反应 Sanger测序法 克拉霉素耐药性 

分 类 号：R372[医药卫生—病原生物学]