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【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。
【Objective】 A TaqMan-MGB probe technique was established for the rapid detection of Borghoff von Wiener-Bartonella species with a high level of specificity and sensitivity. 【Method】 On the basis of sequence-specific amplified region (SCAR) markers, primers and primers were designed according to the unique gene sequence of Bosenhoff von Wiedenbergia to optimize the amplification reaction Annealing temperature, reaction concentration of probe and primer; analyzing the specificity, sensitivity and repeatability of the method; drawing a standard curve to evaluate amplification efficiency and stability of the PCR reaction. 【Result】 The TaqMan-MGB probe designed in this study was level-specific. The minimum detection limit was 11 copies per PCR reaction. The coefficient of variation (CV) within and between groups were 0.12% -0.70% and 0.14 % -0.55%, within the allowable range; the calibration curve has good linearity (R2 = 1) and high amplification efficiency (E = 104.7%). 【Conclusion】 The fluorescent quantitative PCR method based on TaqMan-MGB probe technology established in this study can detect the Borghoff species of Von Wiener Bartonella in a species-specific and highly sensitive manner, Series of diseases early rapid diagnosis, monitoring and epidemiological investigations provide an effective means of research.