Development And Application Of LAMP For Detection Crypsporidium

Cryptosporidiosis is an important zoonotic parasitic diseases caused by Cryptosporidium, and the mammals, birds, fish, reptiles and other animals can cause diarrhea or digestive tract symptoms, it is more serious harm for patient with immune deficiency or inhibition, even have life-threatening. There is more and more environmental pollution with the rapid industrialization and urbanization, the animal or human faeces containing Cryptosporidium oocysts can contaminate food or water, so there is the risk for human or animal infected with Cryptosporidium. Therefore, we need to develop more suitable detection methods for animal faeces or environmental samples. The purpose of this study is to establish a simple, fast, high specificity, high sensitivity molecular methods for detection Cryptosporidium oocysts in the faeces.The study designed2loop primers based on the4specific primers recognized the18S rRNA gene of C. parvum, the method of Loop-mediated isothermal amplification(LAMP) was developed and optimized. The results showed that the process of95℃for denatured and80℃for enzyme inactivation were not necessary, the most optimal reaction temperature and time were63℃and60min, the most optimal concentrations of MgSO4、Betaine、dNTP Mixture and Bst DNA polymerase were8mmol/L、0.8mol/L、0.8mmol/L and8U/25μL respectively, the most optimal concentrations of inner and outer primers were1.2μmol/L and0.2μmol/L, the reaction time was shorten to20min after used the loop primers. The method showed good specificity and detected C. parvum by characteristic ladder mark of LAMP, but can’t detect Giardia sp、Entamoeba sp、 Eimeria tenella、Strongyloides sp and double distilled water. The detection limit of LAMP was5×100oocysts/μL, it degraded2order of magnitude(100times) than the detection limit of PCR. The product of LAMP was authenticated by digested, and we got the expectant result. We took the LAMP product in three different concentrations of agarose electrophoresis, founded that the ladder mark of LAMP was more scattering in1.5%agarose, and more easy to survey. Compared4different result decision methods of LAMP, the method of coloration by SYBR Green Ⅰ was good to use for rapid detection at the scene.The research got the conservative fragment from the18s rRNA gene of C. parvum、C. wrairi、C. muris、C. baileyi and C. serpentis by Clustalx1.83software, and designed LAMP primers based on the fragment, the method of detection Cryptosporidium by visual LAMP was developed and optimized. The optimize results of LAMP showed that the most optimal reaction temperature and time were63℃and60min, the most optimal concentrations of MgSO4、Betaine、 dNTP Mixture and Bst DNA polymerase were8mmol/L、10mol/L、0.8mmol/L and8U/25μL respectively, the most optimal concentrations of inner and outer primers were1.6μmol/L and0.2μmol/L, the reaction time was shorten to20min after used the loop primers. The method showed good specificity and detected C. parvum、C. baileyi、C. muris、C. andersoni、 Cryptosporidium avian genotype V by characteristic ladder mark of LAMP, and the color was (bright)green after coloration by SYBR Green I, but can’t detect Giardia sp、Entamoeba sp、 Eimeria tenella、Strongyloides sp and double distilled water, they had not the characteristic ladder mark of LAMP, and the the color was orange. The detection limit of LAMP was5×101oocysts/μL, it degraded1order of magnitude(10times) than the detection limit of PCR. The product of LAMP was authenticated by digested, and we had the expectant result. The study did systematic and detailed optimization for using SYBR Green I also, we got the results that the most optimal concentrations and amount of SYBR Green I was1000×and1μL, surveyed the color by naked eye directly was the best method. We also innovated a method for adding the dye, the color reaction not needed open the tube, the method was economical, practical, and very suitable for visual result decision of LAMP. The detection limits of SYBR Green I coloration and agarose electrophoresis for LAMP product were similar. Preservative samples of our laboratory were detected for verifying the feasibility of this LAMP method, the detection sensitivity of this method was similar with Nested-PCR.