摘要
目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性和80℃酶失活过程,其最佳反应温度和时间是63℃和60min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8mol/L、8mmol/L、0.8mmol/L、8U/25μL,内外引物浓度分别为1.2μmol/L、0.2μmol/L,添加环引物后体系反应时间缩短为20min。对贾第虫、肠阿米巴、柔嫩艾美耳球虫、类圆线虫检测为阴性。该方法简便、快速,无需特殊设备。结论和常规PCR方法比较,LAMP检测隐孢子虫的灵敏度提高了100倍。该方法的建立为野外和临床隐孢子虫的快速检测提供技术手段。
Loop-mediated isothermal amplification（LAMP） by using SYBR Green I as chromogenic agent was developed and optimized in order to detect C.parvum rapidly.Two loop primers were designed based on the 4 specific primers which recognized the 18S rRNA gene of C.parvum.The process of 95 ℃ thermal denaturation and 80℃ enzyme inactivation were cut out and its＇ most optimal reaction temperature and time were 63 ℃ and 60 min.The best optimum concentrations of Betaine,MgSO4,dNTP Mixture and Bst DNA polymerase were 0.8 mol/L,8 mmol/L,0.8 mmol/L and 8 U/25 μL,respectively.The most optimal concentrations of inner and outer primers were 1.2 μmol/L and 0.2 μmol/L,respectively,and the reaction time was shorten to 20 min after using the loop primers.The method could not detect Giardia sp,Entamoeba sp,Eimeria tenella,and Strongyloides sp,showing good specificity.There were several advantages to the method.Besides being simple and fast to operate,it can be used without special equipment.Compared with traditional PCR method,it results in an approximately 100-fold increase in sensitivity,which could be applied in the clinical field for rapid detection of Cryptosporidium in the near future.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第12期1195-1201,共7页
Chinese Journal of Zoonoses
基金
河南省科技厅重点科技攻关项目(No.92102110138)
国家重大传染病专项-重要寄生虫病检测和监测技术研究(No.2008ZX10004-011)
国家自然科学基金项目(No.31172311)
国家科技重大专项(No.2012ZX10004-220)联合资助~~
关键词
微小隐孢子虫
环介导等温扩增
检测
PCR
Cryptosporidium parvum
loop-mediated isothermal amplification（LAMP）
detection
PCR
