幽门螺杆菌毒力基因数字PCR检测体系的建立  

Establishment of droplet digital PCR detection system for Helicobacter pylori virulence genes

作　　者：范宏博 蔡永洪 李月玥 梁志坚 张力玲 马婷婷 胡松青 胡良勇 FAN Hongbo;CAI Yonghong;LI Yueyue;LIANG Zhijian;ZHANG Liling;MA Tingting;HU Songqing;HU Liangyong(South China University of Technology,Guangzhou Guangdong,510630,China;Guangzhou Institute of Measurement and Testing Technology)

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

出　　处：《质量安全与检验检测》2024年第1期36-41,共6页QUALITY SAFETY INSPECTION AND TESTING

基　　金：广东省重点领域研发计划项目(2020B0404010002)。

摘　　要：建立幽门螺杆菌(Helicobacter pylori)毒力基因数字PCR检测体系,为Hp诊疗提供一个精准、灵敏的毒力定量检测方法。根据Hp毒力基因vacA和cagA的序列设计特异性引物和探针,采用质控菌评估检测特异性;以梯度法设置引物浓度和退火温度,对反应体系及反应条件进行优化;以梯度稀释的DNA为模板,评估检测灵敏度;采用不同浓度模板开展重复性检测,评估检测精密度。结果显示,所设计的引物和探针可特异性地检测vacA和cagA基因,不受大肠杆菌等细菌干扰。vacA和cagA基因的最佳反应温度分别为55.0℃和57.9℃,最佳引物工作浓度分别为650 nmol/L和550 nmol/L,线性分析的拟合度R^(2)分别为0.9991和0.9995,不同浓度样品的变异系数(CV)值均小于10%。本研究建立的Hp毒力基因vacA和cagA数字PCR检测体系具有特异、灵敏和重复性好等优点,可为Hp病理诊疗和科学研究提供精准、可靠的技术支持。This study aimed to establish the digital PCR system for Helicobacter pylori(Hp)virulence genes detection,providing an accurate and sensitive quantitative detection method for Hp diagnosis.Specific primer and probe was designed based on the Hp vacA and cagA 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 and system.The sensitivity was detected using gradient diluted DNA as templates.The accuracy was detected using different concentration templates with multiple repeated tests.The results showed that the designed primers and probes could specifically detect Hp vacA and cagA genes without the interference from E.coli and other bacteria.The optimal reaction temperature for vacA and cagA was 55.0℃and 57.9℃,the optimal primer concentration was 650 nmol/L and 550 nmol/L,the linear fitting degree R^(2) was 0.9991 and 0.9995,and the CV values of samples with different concentrations were less than 10%,respectively.The Hp digital PCR detection for system vacA and cagA established in this study had the advantages of high specificity,sensitivity,and repeatability,which could provide accurate and reliable technical support for Hp pathological detection and scientific research.

关 键 词：幽门螺杆菌 VACA CAGA 数字PCR 精密度 重复性 

分 类 号：Q93-332[生物学—微生物学]