摘要
目的 研究利用环介导等温扩增(loop-mediated isothermal amplification,LAMP)结合核酸试纸条技术建立艰难梭菌(Clostridium difficile,CD)的快速检测方法,为艰难梭菌检测提供一种快速检测的方法。方法 收集2016年1月至2017年12月杭州市临安区第一人民医院临床感染性腹泻患者的粪便标本201份。根据艰难梭菌毒素A基因特异序列设计引物,选择6条最佳引物,用LAMP环介导等温扩增曲线、钙黄绿素目视法和核酸试纸条测试实验结果,再优选反应体系,最后检测粪便中的艰难梭菌。结果 LAMP法能在60℃条件下扩增1h检出艰难梭菌,对1株艰难梭菌和7株非艰难梭菌检测,只有艰难梭菌能检出阳性扩增,设计的引物具有良好的特异性。脱氧核糖核酸(deoxyribo nucleic acid,DNA)检出限为0.8pg/μl。结论 LAMP结合核酸试纸条技术快速检测粪便艰难梭菌具有敏感度高、操作简便、特异性强、可视化、方便快捷的特点,适用于艰难梭菌的快速检测。
Objective A loop-mediated isothermal amplification(LAMP) combined with nucleic acid test strip technology was established for rapid detection of Clostridium difficile, provided a certain reference basis for the detection and prevention of Clostridium. difficile. Methods A total of 201 stool samples were collected from patients with clinical infectious diarrhea from January 2016 to December 2017 in the First People’s Hospital of Lin’an District in Hangzhou city. The six best primers based on the specific sequence of gene tcd A gene were designed and selected, and the experimental results were tested by the LAMP curve, calcein visual method and nucleic acid test strips, and then optimize the reaction system. Results The Clostridium difficile could be rapidly and specifically detected by the LAMP method within 1 h at constant temperature of 60℃. One Clostridium difficile was able to be amplified specific fragments of gene tcd A, while 7 strains of non- Clostridium difficile could not, and no false positive or false negative results occurred, the primers had good specificity. The sensitivity of the method was as low as 0.8 pg/μl. Conclusion LAMP combined with nucleic acid test strip technology for the rapid detection method of clostridium difficile in stool that has the characteristics of high sensitivity, simple operation, strong specificity, visualization, convenience and quickness, and is suitable for the rapid detection of Clostridium difficile.
作者
骆益华
吴丽娜
吴俊
赵明
邵飞琴
张伟群
LUO Yihua;WU Lina;WU Jun;ZHAO Ming;SHAO Feiqin;ZHANG Weiqun(Clinical Laboratory,the First Peopleˊs Hospital of Linˊan District,Hangzhou 311300,China;Linˊan District Center for Disease Control and Prevention,Hangzhou 311300,China)
出处
《中国现代医生》
2022年第23期47-50,共4页
China Modern Doctor
关键词
环介导等温扩增
艰难梭菌
钙黄绿素目视法
核酸试纸条
敏感度
特异性
Loop-mediated isothermal amplification
Clostridium difficile
Calcein visual method
Nucleic acid test strip
Sensitivity
Specificity
