刘芳,王彩霞,綦廷娜,吴芳草,文学琴,陈峥宏,崔古贞
LIU Fang,WANG Caixia,QI Tingna,WU Fangcao,WEN Xueqin,CHEN Zhenghong,CUI Guzhen

• 1:贵州医科大学基础医学院微生物学教研室
• 2:贵州省普通高等学校病原生物学特色重点实验室
• 3:贵州医科大学
• 4:贵州医科大学医学检验学院

摘要(Abstract)：

目的:通过对常规菌落PCR方法进行优化,探索提高幽门螺杆菌(H.pylori)菌落PCR扩增效率的方法。方法:提取H.pylori 26695基因组DNA作为阳性对照组,以未经任何处理的H.pylori 26695菌落作为阴性对照组,经煮沸裂解速冻法获得的H.pylori 26695菌落作为试验组,随机选择8个不同的基因(16S r DNA、cag A-U、cag A-D、ice A、ure A、het A、rop N、Hp0792)作为菌落PCR的候选基因进行菌落PCR验证,最后以1%琼脂糖凝胶进行电泳检测。结果:在选择的目标基因中,阴性对照组未扩增出目的条带,试验组与阳性对照组均扩增出目的相应大小的条带;与常规菌落PCR比较,煮沸速冻裂解法能显著提高H.pylori菌落PCR的扩增效率。结论:煮沸速冻裂解法可用于H.pylori的高通量筛选鉴定和分子诊断。
Objective:To improve the PCR amplification efficiency of Helicobacter pylori(H.pylori colonies by optimizing the conventional colony PCR method.Methods:The genomic DNA from H.pylori 26695 strain was extracted as a positive control group,and H.pylori 26695 colonies without any treatment were used as a negative control group.Lysates derived from boiling-freezing H.pylori26695 colonies were used as experimental groups.Eight genes(16 S rDNA,cagA-U,cagA-D,iceA,ureA,hetA,ropN,Hp0792) were randomly selected for colony PCR.PCR products were finally detected by electrophoresis on a 1% agarose gel.Results:Electrophoresis results show that all selected genes could be amplified in positive control and experimental group,however,it could not be amplified in negative control group.Compared to positive control group,all eight selected genes were also wellamplified in experiment group.Conclusion:The boiling-freezing method can be used for high-throughput screening and molecular diagnosis of H.pylori.

关键词(KeyWords)： 螺杆菌,幽门;高通量筛选;分子诊断技术;煮沸速冻裂解法;菌落PCR
Helicobacter pylori;high-throughput screening;molecular diagnosis;boiling-freezing lysis;colony PCR

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31760318,81460314);; 贵州省研究生科研基金立项项目(11348);; 贵州省科技计划项目[黔科合基础(2018)1132、(2019)1441];; 贵州医科大学学术新苗及创新探索项目[黔科合平台人才(2018)5779-17]

作者(Author): 刘芳,王彩霞,綦廷娜,吴芳草,文学琴,陈峥宏,崔古贞
LIU Fang,WANG Caixia,QI Tingna,WU Fangcao,WEN Xueqin,CHEN Zhenghong,CUI Guzhen

DOI: 10.19367/j.cnki.1000-2707.2019.07.004

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