|
Objective: 1, select an efficient, simple and fast, reasonably priced trace fecal DNA extraction kit. 2, Shanghai a porcine Cryptosporidium parasite identification. 3, a preliminary understanding of some children with diarrhea in Zunyi, cancer patients and livestock Cryptosporidium infection and its epidemiological characteristics. Method: 1, using three different extraction kit fecal DNA, by comparing the extracted DNA concentration, purity, extraction rate and nested PCR products were evaluated three kinds of manure and other trace DNA extraction kit. 2, Shanghai a manure samples were modified - acid-fast staining to determine Cryptosporidium infection, measuring oocyst size; 18SrRNA gene DNA was extracted after nested PCR amplification, PCR products were sequenced using Blast and MEGA software for homologous and phylogenetic analysis. 3, Affiliated Hospital of Zunyi Medical College collected pediatric patients with diarrhea and oncology patients, the surrounding towns dogs, cattle, pigs, a total of 582 stool specimens, modified acid-fast staining of Cryptosporidium oocysts, all samples DNA was extracted after 18SrRNA gene nested PCR Amplification. Results: 1, Genmed kit highest concentration of the extracted DNA, PCR products of the brightest bands, but the extraction time is relatively long, the price is relatively high. Day root kit highest purity of extracted DNA, DNA concentration and affordable extraction shortest time. Pik-day kit extracted DNA purity and concentration are not high and the extraction longest time, but the price is the cheapest. 2, Shanghai manure samples were modified - acid-fast staining oocysts were roses red, round, and its size is (4.90 ± 0.83) μm X (4.52 ± 0.73) μm; through 18SrRNA gene analysis, Shanghai swine Cryptosporidium with Brazil a Cryptosporidium pig genotype Ⅱ (Cryptosporidium sp. pig genotype Ⅱ) was 100% homology, phylogenetic tree, both in the same branch. 3, Zunyi all human and animal fecal samples collected by modified acid-fast staining, microscopy Cryptosporidium oocysts were not found; 18SrRNA all samples extracted DNA was amplified by nested PCR were specific fragments. Conclusion: 1, Genmed trace fecal DNA extraction kit most sensitive; day root kit to extract DNA for high purity requirements of molecular biology research. 2, Shanghai swine identified as Cryptosporidium Cryptosporidium pig genotype Ⅱ {Cryptosporidium sp. Pig genotype Ⅱ), 3, through the Zunyi some children with diarrhea, cancer patients and animal feces checked yet found Cryptosporidium infection.
|