Exploration of Loop-mediated Isothermal Amplification(LAMP) for detection of Cryptosporidium parvum

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.