Denaturing gradient gel electrophoresis (DGGE) 法による水中のクリプトスポリジウムの種・遺伝子型判別手法の開発

抄録

A new genotyping method of Cryptosporidium, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing, was developed to genotype Cryptosporidium spp. in river water. The DGGE method could successfully discriminate nine species of Cryptosporidium: C. parvum, C. hominis, C. canis, C. meleagridis, C. felis, C. sp. strain 938, C. andersoni, C. serpentis and C. saurophilum. The sequential combination of the QProbe PCR method for quantification; and the DGGE method and DNA sequencing for genotyping, enabled the simultaneous quantification and genotyping of Cryptosporidium spp. in the same water sample. This method was applied to analyze Cryptosporidium in the Koyama River, a tributary of the Tone River, both for the total concentration of organisms, and for their genotype. Seven Cryptosporidium genotypes (C. andersoni, C. sp. strain 938, C. parvum, C. hominis, C. sp. PG1-26, C. sp. t03, C. sp. t04) were found in 11 positive samples (positive ratio=69%). A bovine specific species, C. andersoni, was found most frequently (7 samples). The genotypes infectious to human accounted for only 16% of the concentration of all genotypes. These results showed that this detection method could provide valuable information on Cryptosporidium in river water both in the quantity and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.