荧光定量PCR快速检测幽门螺杆菌的研究

Real-time quantitative PCR for detection of Helicobacter pylori

目的:建立一种快速检测幽门螺杆菌的荧光定量PCR方法。方法:根据每个幽门螺杆菌有一个拷贝的尿素酶A基因,设计并优化SYBGReen荧光定量PCR条件,评价荧光定量PCR的灵敏度与重复性;收集胃粘膜标本65份,并对其同时进行培养和荧光定量PCR检测。结果:该方法连续8个浓度模板良好线性关系(相关系数,0.997),灵敏度达1拷贝/微升;随机选取两个浓度的样本做重复试验,结果显示具有较高的重复性。65份标本中采用荧光定量PCR方法检出幽门螺杆菌42例(64.62%);细菌培养方法检出31例HP(47.69%)。结论:荧光定量PCR技术能够快速、准确检测幽门螺杆菌,适合于临床检测及科学研究。

Objective：To develop a real-time quantitative（Q） PCR-based assay to detect H.pylori.Methods：The assay to measure ureA gene copy number to detect H.pylori was based on the fact that there is only one copy of the ureA gene per bacterium.Upon optimization of SYBGReen Q-PCR conditions,the parameters of the developed Q-PCR including sensitivity and reproducibility were evaluated.We investigated 65 clinical biopsy samples by the Q-PCR and bacterial culture synchronously.Results：We obtained a standard curve with a linear range（correlation coefficient,0997） across eight logs of DNA concentration.We were able to accurately quantify as few as 1 copy in our assay.Analysis of variance on two logs of DNA concentration clinical biopsy samples randomly selected showed good reproducibility of this assay.Among 65 samples,42 samples were were positive by Q-PCR,Comparison of Q-PCR results with bacterial culture,31 samples were positive for H.pylori by culture.Conclusion：We developed a rapid,sensitive,and real-time Q-PCR method for detecting H.pylori.This technique offers a significant improvement over other available methods for detecting H.pylori in clinical and research samples.