Loop-mediated isothermal amplification (LAMP) by using SYBR Green I as ehromogenie 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 denatnration 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 BstDNA polymerase were 0 .8 mol/L , 8 mmol/L,0.8 mmol/L and 8 U/25μL , 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 .%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18S rRNA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60 min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8 mol/L、8 mmol/L、0.8 mmol/L、8U/25 μL,内外引物浓度分别为1.2 μmol/L、0.2 μmol/L,添加环引物后体系反应时间缩短为20 min.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.
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