Genotyping of Cryptosporidium oocysts in wastewater using a semi-nestd PCR- Direct sequence technique
| Project/Area Number |
16560484
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Civil and environmental engineering
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| Research Institution | School of Environmental Health, Azabu University |
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Principal Investigator |
HIRATA Tsuyoshi Azabu University, School of Environmental Health, Professor, 環境保健学部, 教授 (50005493)
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| Co-Investigator(Kenkyū-buntansha) |
HASHIMOTO Atsushi Anan National College of Technology, Department of Civil Engineering, Assistant Professor, 環境システム学科, 講師 (30332068)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Cryptosporidium / oocysts / 18S rRNA / genotyping / semi-nested PCR / sewage / Cryptosporidium / 遺伝子型 / PCR法 / ダイレクトシークエンス / 河川水 / ダイレクトシーケンス / 塩基配列 |
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| Research Abstract |
Single immuno-fluorescent assay (IFA)-stained Cryptosporidium oocysts were isolated from raw sewage concentrates of a metropolitan sewage treatment plant using glass capillary pipettes under inverted epi-fluorescence microscopy. Oocysts concentration was in the range of 1 to 20 oocysts/L with a geometric mean of 4.2 oocysts/L.Peak concentration was observed in August in 2003, September in 2004 and March and August in 2005. Total number of isolated oocysts amounted to 239 oocysts. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 148 (62%) of 239 oocysts isolated from sewage were positive in the semi-nested PCR and 121 oocysts (51%) were genotyped successfully. Seventy eight oocysts (33%) were identified as C.parvum genotype 1,16 oocysts (7 %) as C.parvum genotype 2,13 oocysts (5%) as C.meleagridis, 6 oocysts (3%) as C.parvum VF383 human isolates, 5 oocysts (2%) as C.sp.pig 1,2 oocysts (1%) of C.sp PG1-26 pig isolates and 1 oocyst (0.4%) as C.parvum CPM1 isolated from mouse. Total number of oocysts suspected to be infectious to human amounted to 84 oocysts, corresponding to 47% of isolated oocysts and 69% of successfully genotyped. The results of this study demonstrate that 18S rRNA based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.
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Report
(3 results)
Research Products
(7 results)
