下水中のCryptosporidium parvumオーシスト濃度と遺伝子型に関する汚染実態調査(平成14年度麻布大学公的研究助成金事業研究成果報告)

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We tried to detect and identify a single Cryptosporidium oocyst in sewage and river water using PCR-RFLP or PCR direct sequence assay. Prior to application to environmental samples, the ability of the PCR to amplify target DNA from one oocyst was examined using C. parvum HNJ-1 strain. Each oocysts isolated using glass capillary was transferred to PCR buffer containing 1% TX-100 within a PCR tube and then subjected to three freeze-thaw cycles. Using two primer sets which amplify portion of the sequence encoding 18S rRNA or poly threonine, PCR amplification was observed in 10 of 10 samples by 18S rRNA based PCR and 47 of 53 samples by poly threonine based PCR. The results demonstrated that PCR amplification of a single oocyst was applicable in purified C. parvum oocyst. Then, we tried to examine the applicability of single oocyst PCR to raw sewage and river water samples. PCR amplification was observed only one of 30 samples (16 samples of 18S rRNA and 14 samples of poly threonine based PCR, respectively) from raw sewage by 18S rRNA based PCR. The PCR product was sequenced and identified as 18S rRNA gene fragment of C. parvum isolate HCTX8 (genotype 2) by BLAST. Trials were also carried out with a single oocyst from river water by poly threonine based PCR. Three of 10 samples from river water were positive. The three PCR products were analyzed by RFLP, and all RFLP patterns showed similarity to the RFLP pattern of C. parvum genotype 2. In addition, two of the three products were sequenced successfully and identified as poly threonine gene fragment of C. parvum genotype 2 by BLAST.
麻布大学の論文

下水中のCryptosporidium parvumオーシスト濃度と遺伝子型に関する汚染実態調査(平成14年度麻布大学公的研究助成金事業研究成果報告)

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