小鼠胃组织中幽门螺杆菌SYBR GreenⅡ实时定量PCR检测方法的建立及验证  

Development and verification of a SYBR GreenⅡreal-time PCR assay for Helicobacter pylori in stomach of mice

作　　者：陈敬[1] 刘钰[1] 汤重发 刘梅影[1] 魏博[1] 

机构地区：[1]北京生物制品研究所有限责任公司,北京101111

出　　处：《中国生物制品学杂志》2016年第12期1308-1315,共8页Chinese Journal of Biologicals

摘　　要：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1×10^7-1×10^2拷贝/μl范围内线性良好,相关系数r^2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 develop and verify a SYBR Green Ⅱ real time quantitative PCR assay for Helicobacter pylori（Hp）in the stomach of mice in order to evaluate the immune effect of vaccine. Methods Based on the property that the genes encoding Urease B subunit（Ure B）were highly conserved in various Hp strains, specific primers were designed and the condition for PCR was optimized. The developed SYBR Green Ⅱ real time quantitative PCR assay was verified for specificity, precision, accuracy and detection limit. Results The target gene fragment was amplified from the positive samples by using the designed primers, and the existence of other bacteria in the stomach of mice showed no influence on the test result. Standard curve of the assay showed good linearity within a DNA concentration range of 1 × 10^7- 10^2 copies / μl, with a correlation coefficient（r^2 value）of more than 0. 995 and an amplification efficacy of 90% - 110%. The method showed high accuracy and precision in dectection of colonized H. pylori in the stomach of mice, of which the recovery rate was 96. 25% - 101. 42 % and the relative standard deviations（RSDs）were less than 6% in both intra- and inter-assays. All the RSDs of detection results of Hp culture at high, moderate and low concentrations with or without mouse stomach tissue were less than 6% in both intra- and inter-assays, indicating high precision. The recovery rate of the method was more than 80%, which was consistent with that of counting in plate. However, the sensitivity of the former was significantly higher than that of the latter. Conclusion The developed SYBR Green Ⅱ real time quantitative PCR assay was rapid, effective and simple, with good specificity, wide linear range and high reproducibility, which might be used for quantitative detection of Hp colonization in murine stomach and for evaluation of the effect of immunes response on bacterial colonization.

关 键 词：幽门螺杆菌 SYBR Green实时定量PCR 尿素酶B亚基 

分 类 号：R378.99[医药卫生—病原生物学] Q789[医药卫生—基础医学]