2004 Fiscal Year Final Research Report Summary
A novel PCR method for identifying bacteria and plankton DNA in cases of death by drowning
| Project/Area Number |
15390216
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | FUKUSH1MA MEDICAL UNIVERSITY |
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Principal Investigator |
HIRAIWA Kouichi Fukushima Med. Univ., Schl. of Med., Professor, 医学部, 教授 (60124616)
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| Co-Investigator(Kenkyū-buntansha) |
KURISAKI Emiko Fukushima Med. Univ., Schl. of Med., Assistant professor, 医学部, 講師 (30106356)
ABE Sumiko Fukushima Med. Univ., Schl. of Med., Assistant professor, 医学部, 講師 (50136975)
MIZUSAWA Ikubumi Fukushima Med. Univ., Schl of Med., Research assistant, 医学部, 助手 (40192356)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Drowning / PCR / Bacteria / Plankton |
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| Research Abstract |
Neither an acid digestion method nor a PCR method for identifying plankton is useful in cases of death by drowning if inhaled water contains no planktons. So we developed a new PCR method for identifying bacteria and the usefulness of identifying bacterial genes for diagnosing drowning was evaluated.
We examined the bacteria species in blood in the heart and in throat and tracheal swabs obtained from autopsy cases using sterilized instruments. Identification of bacteria in the samples was carried out by the conventional culture method. In throat and trachea swabs obtained from non-drowning cases, Streptococcus (Strep.) and Staphylococcus species were identified, but no bacteria were identified in blood samples. In cases of death by drowning, Strep., Enterobacter and Aeromonas species were identified in throat, trachea and also blood samples. It was thought that the bacteria species that normally localized in the throat and trachea invade into blood through the respiratory tract as well as species localized in water in drowning cases.
Based on these results, we designed primer pairs for membrane protein gene of Strep. salivarius (SL1), which is a common bacteria in the throat, and for ATCC 7966 DNA gyrase subunit B gene of Aero. hydrophila (AH1 and AH2), which has been isolated from various water samples. Both of the cultured strains were obtained from RIKEN BioResource Center. Genomic DNA of both bacteria species was extracted using an AquaPure Genomic DNA kit (BioRad). Employing target DNA as a template, the expected lengths of PCR products amplified with SL1, AH1 and AH2 were 152, 212 and 201 bp, respectively. No PCR product was amplified from human or non-target DNA. With SL1, up to 100 pg of Strep.-DNA was identified, and up to 1 μg of Aero.-DNA was detectable with All or AH2 in each sample mixed with 1 μg of human DNA. The development of this PCR method is a step toward the establishment of a reliable method for diagnosing drowning.
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