Species Identification Of Cryptoporidium Derived From A Pig In Shanghai & Preliminary Investigation Of Cryptosporidium Infection Of Humans And Animals In Zunyi

Objective:1.To Choose an efficient,simple,economic kit for Tiny DNA extraction from stool.2.Identify species of Cryptosporidium derived from a pig in Shanghai. 3.Preliminary Investigation the infective status and epidemiological characteristics of cryptosporidiosis from patients (children with diarrhea, patients with phymatosis) and livestocks in Zunyi.Methods:1.Extract genomic DNA from fecal samples by the three stool tiny DNA extraction kits.Three kits were evaluated by comparison of the extracted DNA of the concentration, purity, extraction rate and the nested PCR product.2.The oocyst of Cryptosporidium from a pig fecal sample in Shanghai was detected by the modified acid-fast staining,the Size of oocyst was measured. Genomic DNA was extracted from fecal sample of pig. The partial 18SrRNA gene were amplified by nested PCR, and sequenced. Homology and phylogeny were analyzed with Blast and MEGA software.3.The oocysts of Cryptosporidium in 582 feces samples specimens from the patients in Affiliated Hospital of Zunyi Medical College (children with diarrhea, patients with phymatosis) and the animals in countrysides of zunyi(dogs,cattles,pigs) were examined by modified acid fast stain.Genomic DNA were extracted from all fecal specimens. The partial 18SrRNA gene were amplified by nested PCR.Results:1.The concentration of DNA with Genmed kit was the highest, PCR product band was the brightest, but the extraction time was relatively long and the price was the highest.The purity of DNA with TIANGEN kit was the highest, extraction time was the shortest, the concentration of DNA was moderate.The price of Beyotime kit was the cheapest, but purity and concentration of DNA were the lowest, and the extraction time was the longest.2. The Size of oocyst from a pig fecal sample in Shanghai was oval in shape and (4.90±0.83)μm x (4.52±0.73)μm in size by the modified acid-fast staining. The results suggested that the sequence identity of Cryptosporidium from a pig fecal sample in Shanghai with Cryptosporidium sp. pig genotype II from Brazil appeared to be 100%, as demonstrate by 18S rRNA, they were branched in a same clade of the phylogeny tree.3.The oocysts of Cryptosporidium from the feces samples of the patients and livestocks in Zunyi could not be detected by the modified acid-fast staining. The genomic DNA of all samples amplified by the 18S rRNA gene nested PCR,but all were not amplified specific fragments.Conclusion:1.The Genmed kit extracted stool micro DNA was the most sensitive for stool samples,extraction of DNA with TIANGEN kit required a higher purity DNA suitable for molecular biological research.2. The Cyptosporidium from a pig fecal sample in Shanghai was identified as Cryptosporidium sp. pig genotype II.3. Cryptosporidiosis had not been found among children with diarrhea, patients with phymatosis and the livestocks in Zunyi.