刘正美,周建奖,赵艳,熊林,龙妮娅,谢渊
LIU Zhengmei,ZHOU Jianjiang,ZHAO Yan,XIONG Lin,LONG Niya,XIE Yuan

• 1:贵州医科大学
• 2:贵州医科大学分子生物学重点实验室

摘要(Abstract)：

目的:建立一种幽门螺杆菌(H.pylori)临床菌株的分离培养的方法。方法:取临床胃黏膜组织标本88株,接种于10%绵羊全血的哥伦比亚选择性培养基上,37℃、5%O2、10%CO2、85%N2培养3~5 d后,挑取血平板上透明细小可疑菌落进行革兰氏染色和尿素酶试验,对阳性菌落进行扩大纯培养,提取细菌DNA,PCR扩增H.pylori 16sRNA、Cag A基因,电泳并进行测序鉴定。结果:成功从临床胃黏膜组织中分离培养出H.pylori,88例胃黏膜组织标本中分离培养并鉴定出H.pylori 19株,阳性率为22%;对16sRNA及Cag A基因进行扩增,电泳可见目的条带,并对16sRNA扩增产物测序,与H.pylori国际标准菌株26695比对,同源性为95%~99%。结论:37℃、5%O2、10%CO2、85%N2条件下10%绵羊全血的哥伦比亚选择性培养基上能成功分离培养出H.pylori临床株。
Objective: To isolate and culture the clinical strains of Helicobacter pylori( H. pylori)from patients in the Affiliated Hospital of Guizhou Medical University,and to master the methods of isolation,culture and identification of clinical isolates. Methods: 88 samples of specimens were collected from clinical gastric mucosal tissue and inoculated on columbia selective medium with 10%sheep blood,then cultured on the condition of 37 ℃,5% O2,10% CO2,85% N2 for 3 ~ 5 days.The transparent small suspicious colonies on blood agar were picked out to undergo Gram staining and rapid urease test. The positive colonies were expanded by pure culture. DNA of bacterial was extracted and 16 sRNA of H. Pylori was amplified by PCR. Electrophoresis and sequencing were conducted for identification. Results: 19 strains of H. Pylori from 88 samples of specimens collected from clinical gastric mucosal tissue were successfully isolated,cultured and identified. The positive rate was 22%.16 sRNAand Cag A of H. Pylori were amplified by PCR and their electrophoresis strips were clearly visible. The sequenced 16 sRNA amplification products were compared with H. pylori international standard strain 26695,and their homology was 95% ~ 99%. Conclusion: H. pylori clinical strains are successfully isolated and cultured,and the method of isolation,culture and identification of H. pylori clinical isolates was skillfully mastered.

关键词(KeyWords)： 幽门螺杆菌;分离培养;临床菌株;革兰染色;CagA基因
Helicobacter pylori;isolation and culture;clinical strains;Gram stain;CagA gene

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(No.81260303)

作者(Author): 刘正美,周建奖,赵艳,熊林,龙妮娅,谢渊
LIU Zhengmei,ZHOU Jianjiang,ZHAO Yan,XIONG Lin,LONG Niya,XIE Yuan

DOI: 10.19367/j.cnki.1000-2707.2016.04.010

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