小家鼠螺杆菌TaqMan-qPCR检测方法的建立

doi:10.3969/j.issn.1674-5817.2017.05.007

实验动物与比较医学 ›› 2017, Vol. 37 ›› Issue (5): 378-382.DOI: 10.3969/j.issn.1674-5817.2017.05.007

小家鼠螺杆菌TaqMan-qPCR检测方法的建立

伍妙梨, 朱余军, 饶丹, 袁文, 王静, 丛锋, 黄韧, 陈梅丽, 郭鹏举

广东省实验动物监测所, 广东省实验动物重点实验室, 广州510663

收稿日期:2017-03-23 出版日期:2017-10-25 发布日期:2017-10-25
作者简介:伍妙梨(1987-),女,硕士,研究方向:预防兽医学。E-mail:983671845@qq.com
基金资助:
广东省省级科技计划项目(2013B060400028;2014A070705003;2014B070706006)

Development of TaqMan-qPCR for Detection of Helicobacter muridarum

WU Miao-li, ZHU Yu-jun, RAO Dan, YUAN Wen, WANG Jing, CONG Feng, HUANG Ren, CHEN Mei-li, GUO Peng-ju

Guangdong Laboratory Animals Monitoring Institute, Guangdong Key Laboratory of Laboratory Animals, Guangzhou 510663, China

Received:2017-03-23 Online:2017-10-25 Published:2017-10-25

摘要/Abstract

摘要： 目的建立敏感性高、特异好的小家鼠螺杆菌(Helicobacter muridarum) TaqMan-qPCR检测方法。方法选择小家鼠螺杆菌保守基因GyrB设计特异性引物进行PCR扩增,并构建质粒标准品GyrB-19T。通过反应体系及条件的优化,建立小家鼠螺杆菌TaqMan-qPCR检测方法,并对GyrB-19T标准品进行定性及定量分析,评估该方法的灵敏度、特异性及重复性。结果所建立的qPCR检测方法,质粒标准品浓度在2×10²~2×10⁸拷贝/μL范围内表现出较好的线性相关性,所得标准曲线的斜率为-3.57,相关系数为0.996,检测灵敏度达到200拷贝/μL。结论所建立的小家鼠螺杆菌TaqMan-qPCR方法具有特异性好、敏感性高、稳定性强等特点,适用于小家鼠螺杆菌的定量及定性分析。

关键词: 小家鼠螺杆菌, TaqMan-qPCR

Abstract: Objective To establish the sensitive and specific TaqMan-qPCR method for detection of Helicobacter muridarum. Method The specific primers were designed according to the Helicobacter muridarum gyrase B(GyrB) gene, specific fragment was amplified by PCR, and cloned into pMD-19T vector to construct the recombinant plasmid GyrB-19T, which was used as standard DNA of this qPCR method. The qPCR system was optimized and the sensitivity, specificity, repeatability and quantitative range of this method were evaluated. Result The quantitative standard curve from 2×10⁸copies/well to 2×10²copies/well of serial diluted plasmid DNAs showed that they had good liner correlation, the slope of the standard curve was -3.57, R²>0.996, and the lowest detection limit reached 2×10²copies/well. Conclusion This qPCR method showed high sensitivity, specificity and stability, which will be utilized for qualitative and quantitative detection of Helicobacter muridarum.

Key words: Helicobacter muridarum, TaqMan-qPCR

中图分类号:

S855.3Q95-33

伍妙梨, 朱余军, 饶丹, 袁文, 王静, 丛锋, 黄韧, 陈梅丽, 郭鹏举. 小家鼠螺杆菌TaqMan-qPCR检测方法的建立[J]. 实验动物与比较医学, 2017, 37(5): 378-382.

WU Miao-li, ZHU Yu-jun, RAO Dan, YUAN Wen, WANG Jing, CONG Feng, HUANG Ren, CHEN Mei-li, GUO Peng-ju. Development of TaqMan-qPCR for Detection of Helicobacter muridarum[J]. Laboratory Animal and Comparative Medicine, 2017, 37(5): 378-382.

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小家鼠螺杆菌TaqMan-qPCR检测方法的建立

Development of TaqMan-qPCR for Detection of Helicobacter muridarum

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