The Exploratory Development Of The FTA/PCR Method To Detect The Cryptosporidium Ocysts In Feces

Cryptosporidium spp.is an important identified gastro-intestinal zoonotic protozoas worldwide that can be transmitted via either direct contact or ingestion of contaminated food or water.Currently Cryptosporidium detection methods include stool examination,immunological and molecular biological methods and so on.Represented by PCR molecular biology methods need to be relatively clean nucleic acid test sample material as DNA template of the PCR reaction.So obtain high purity of DNA samples in the PCR reaction is essential.FTA card is a commercial product based on technology of dry blood spots,worldwide used in most kinds of nucleic acid substances.Research shows that current detection of Cryptosporidium is carried out only in waterborne Cryptosporidium.This research will establish the FTA/PCR method for detection of Cryptosporidium in the feces with dairy manure as an example.And compared with sold commercial fecal DNA extraction Kit for the DNA extraction effect.The method is a groundwork for the detection of Cryptosporidium oocysts in feces.Continuing to use the subculture method of Cryptosporidium parvum founded by our laboratory,by removing manure impurity,eliminating fat,ether discontinuous sucrose density gradient centrifugation and purified by cesium chloride density gradient centrifugation method,I got totaling 1.56x108 Cryptosporidium oocysts.And I identified the subtype of Cryptosporidium oocysts is IId A19G1 via the GP60 gene.Based on Cryptosporidium SSU r RNA genetic loci,using the Cryptosporidium oocysts obtained previous by FTA/PCR method to detect Cryptosporidium after purification.The minimum detection limit is 1x101oocyst/m L by the FTA/PCR method.In all of the test objects included C.parvum IId A19G1,and C.parvum IId A15G2,C.andersoni,and Cyclospora,Giardia,Coccidia,Based on Cryptosporidium specificity primer only detect C.parvum IId A19G1,C.parvum IId A15G2,C.andersoni.Based on SSU r RNA genetic loci,fecal samples from pretreatment,purifing DNA template,optimizing of PCR primers,optimizing the PCR reaction conditions,I established and optimized the FTA/PCR method to detect Cryptosporidium oocysts in feces.Through the feces samples saturated sucrose floating,cleaning test,design specificprimer sequences Through a series of experiments such as saturated sucrose flotation,cracking and chemical reagents and ion detergent to clean,designed specific primers sequence,we identified in 5% SDS(sodium dodecyl sulfate)solution boiling FTA card for5 min,combined with Whatman FTA card provided by the company cleaning method to conventional elution steps.Based on the SSU r RNA gene fragment length 540 bp and250bp of Cryptosporidium primer,meanwhile joined the BSA(protein calf serum)in the nested PCR reaction of the FTA cleaning method and PCR amplification procedure.With the above methods,the detection minimum limit in stool is 1x101 oocysts/g feces via the FTA/PCR method.Specific detection of Cryptosporidium oocysts for display-specific primers amplified Cryptosporidium purposes only,the sequencing results and standard sequence alignment display of Cryptosporidium,the homology of 100%.The 72 dairy calves fecal samples on a dairy farm were randomly collect from zhongmou area in zhengzhou city.The DNA of Cryptosporidium oocysts were extracted from the feces sample with the commercialization of fecal DNA extraction kit and FTA card.And were detected for the molecular biology with the nested PCR.The results showed that 18 samples were positive Cryptosporidium with the overall prevalence being25.35%(18/72)based on SSU r RNA gene through the FTA/PCR method.Which brings into correspondence with the detection result of Commercial test fecal DNA extraction kit.In conclusion,This research devoted to establishment and optimization of the FTA/PCR method for the detection of Cryptosporidium oocysts in feces.And we proceeded the established method for preliminary application.The results showed that the specificity and sensitivity of this method satisfied the test requirements for detection of Cryptosporidium oocysts in feces.The FTA/PCR method to more convenient,efficient and safe detection and prevention and development of Cryptosporidium,has a certain public health significance The establishment of the FTA/PCR method is helpful to a more convenient,quick,efficient,safe detection.The method can prevent the occurrence of cryptosporidiosis,which has a certain public health significance.