胆汁螺杆菌实时荧光定量PCR检测方法的建立

目的 建立快速、敏感、特异的胆汁螺杆菌(Helicobacter bilis,H.bilis)TaqMan探针实时荧光定量PCR检测方法,对胆汁螺杆菌进行定量检测。方法 PCR扩增H.bilis保守基因P17序列全长ORF435 bp,进行TA克隆,构建质粒标准品pMD-HBP17。通过对pMD-HBP17标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量PCR方法的灵敏度、特异性及重复性;用所建立的qPCR方法检测77份临床样品,并与普通PCR的检测结果作比对。结果 所建立的qPCR检测方法,质粒DNA浓度在10⁸~10¹拷贝之间表现出较好的线性和相关性,所得标准曲线的斜率为-3.46,相关系数为0.999,检测灵敏度达到20个拷贝,对77份临床样品的检出率为14.3%,较普通PCR(7.8%)的检出率高。结论 建立的H.bilis qPCR检测方法特异性好、敏感性高、稳定性强,可用于胆汁螺杆菌的定量及定性检测。

Detection of Helicobacter bilis using quantitative real-time PCR with Taqman probe

Objective To develop a rapid, sensitive and specific assay based on TaqMan probe real-time PCR to quantitate Helicobacter bilis(H.bilis). Method A 435 bp specific fragment of H.bilis P17 gene was amplified by PCR, then cloned into pMD19-T vector to construct a recombinant plasmid pMD-HBP17, which was used as standard DNA of this qPCR method. The qPCR system was optimized by using serial dilution of standard plasmid. The sensitivity, specificity, repeatability and quantitation range of this method were evaluated. The established method was used to detect 77 clinical samples. Result The quantitative standard curve from 10⁸ copies/ well to 10¹ copies/well of serial diluted plasmid DNAs showed that they had good linear correlation, the slope of the standard curve was -3.46, R²>0.999, and the lowest limit reached 2×10¹ copies/well. The positive rate of H.bilis detected by qPCR was 14.3% which is higher than detected by PCR(7.8%). Conclusion ThisqPCR method showed high sensitivity, specificity and stability and will be utilized for qualitative and quantitative detection of H.bilis.