幽门螺杆菌ureC和23SrDNA数字PCR检测体系的建立  

Establishment of Digital PCR Detection System for Helicobacter pylori ureC and 23S rDNA

作　　者：范宏博 胡良勇 胡松青 FAN Hongbo;HU Liangyong;HU Songqing(Department of Science and Technology Development,South China University of Technology,Guangzhou 510630,China;Guangzhou Institute of Measurement and Testing Technology,Guangzhou 510700,China)

机构地区：[1]华南理工大学食品科学与工程学院,广州510630 [2]广州计量检测技术研究院,广州510700

出　　处：《生物技术进展》2024年第5期868-874,共7页Current Biotechnology

基　　金：广东省重点领域研发计划项目(2020B0404010002)。

摘　　要：为了建立幽门螺杆菌(Helicobacter pylori,Hp)ureC和23S rDNA基因的数字PCR检测体系,为Hp检测提供一个可靠的参考方法,根据Hp ureC和23S rDNA基因序列,设计了特异性引物和探针,采用质控菌评估了检测特异性;通过设定引物浓度和退火温度的梯度,对检测参数进行了优化;通过梯度法稀释DNA模板,评估了检测灵敏度;使用不同浓度模板开展了重复性检测,以评估检测精密度。经过评估,该检测方法能够特异性地用于ureC和23S rDNA基因的检测,并且不受大肠杆菌等细菌干扰。ureC和23S rDNA的最佳反应温度均为55.8℃,而最佳引物浓度分别为550和650 nmol·L^(-1),通过线性分析,两种基因的拟合度R^(2)值分别高达0.9991和0.9997,显示了良好的线性关系。此外,不同浓度样品的变异系数(CV)均小于10%,说明该检测方法具有高度的重复性。研究成功建立了Hp ureC和23S rDNA基因数字PCR检测体系,具备高度的特异性、灵敏度和重复性,为幽门螺杆菌的检测、诊断和科学研究提供了一个可靠的检测方法和技术支持。This study aimed to establish the digital PCR system for detection of Helicobacter pylori(Hp)genes ureC and 23S rDNA,providing an reliable reference detection method for Hp diagnosis.Specific primers and probes were designed based on the Hp ureC and 23S rDNA sequences,and quality control bacteria was used to conduct specificity testing.The gradient method was used to set the primer concentrations and annealing temperatures to optimize the reaction conditions.The sensitivity was detected using gradient diluted DNA as templates.The accuracy was detected using different concentration templates with multiple repeated tests.The designed primers and probes could specifically detect Hp ureC and 23S rDNA without the interference from E.coli and other bacteria.The optimal reaction temperature for ureC and 23S rDNA was both 55.0℃,the optimal primer concentration was 550 and 650 nmol·L^(-1),the linear fitting degree R^(2) was 0.9991 and 0.9997,and the CV values of samples with different concentrations was less than 10%.The Hp digital PCR detection system for ureC and 23S rDNA established in this study has the advantages of high specificity,sensitivity,and repeatability,which can provide accurate and reliable technical support for Hp pathological detection and scientific research.

关 键 词：幽门螺杆菌 ureC 23S rDNA 数字PCR 

分 类 号：Q789[生物学—分子生物学] R446.5[医药卫生—诊断学]