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目的建立小鼠胃组织中幽门螺杆菌SYBR GreenⅡ实时定量PCR检测方法,并进行验证,以进一步应用于疫苗免疫效果评价。方法根据幽门螺杆菌尿素酶B亚基(urease B,Ure B)在各菌株中相对保守的特性设计引物,优化其扩增条件,建立幽门螺杆菌SYBR GreenⅡ实时定量PCR检测方法,并对方法的专属性、精密性、准确性及检测限进行验证。结果所设计的引物在阳性样本中可特异性地扩增出目的基因片段,且小鼠胃部中其他菌体的存在不影响反应结果的特异性。标准品质粒DNA模板含量在1×107~1×102拷贝/μl范围内线性良好,相关系数r2大于0.995,扩增效率在90%~110%之间。应用于小鼠胃组织幽门螺杆菌检测,具有较好的准确性和精密性,回收率为96.25%~101.42%,试验内和试验间相对标准偏差(relative standard deviation,RSD)均小于6%。用该方法检测高、中、低浓度的Hp菌液、并模拟定量Hp菌株在小鼠胃组织中的状态,试验内及试验间RSD均小于6%,精密度良好。其回收率大于80%,与平皿计数法一样,但灵敏度显著高于平皿计数法。结论 SYBR GreenⅡ实时定量PCR法快速有效,专属性良好,线性范围广,可重复性好,使用方便,可用于定量检测幽门螺杆菌在小鼠胃部组织中的定植,从而进一步应用于评估免疫反应对细菌定植的影响。
Objective To establish a real-time quantitative PCR assay for detection of Helicobacter pylori in gastric tissues of mice and to validate it for further evaluation of vaccine immunization efficacy. Methods Based on the relatively conservative characteristics of urease B (Ure B) in each strain, the primers were designed and their amplification conditions were optimized. The detection method of SYBR Green Ⅱ real-time quantitative PCR was established, Specificity, precision, accuracy and detection limits to verify. Results The designed primers could specifically amplify the target gene fragment in the positive samples, and the presence of other bacteria in the stomach of mice did not affect the specificity of the reaction results. The content of standard plasmid DNA template was linear in the range of 1 × 107 ~ 1 × 102 copies / μl, the correlation coefficient r2 was greater than 0.995, and the amplification efficiency was between 90% and 110%. The method was applied to the detection of Helicobacter pylori in gastric tissues of mice with good accuracy and precision. The recovery rate was 96.25% ~ 101.42%. The relative standard deviations (RSDs) were both less than 6%. The method was used to detect Hp bacteria in high, medium and low concentrations and to simulate the state of Hp strains in the stomach of mice. The intra-and inter-experimental RSD were less than 6% and the precision was good. The recovery was greater than 80%, same as the plate count method, but the sensitivity was significantly higher than the plate count method. Conclusion SYBR Green Ⅱ real-time quantitative PCR method is rapid and effective, with good specificity, wide linear range, good repeatability, easy to use, and can be used to quantitatively detect the colonization of H. pylori in the stomach tissue of mice so as to further evaluate the immune response Impact on bacterial colonization.