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Studies on Forensic Species Identification by Fluorescent Multiplex PCR

森, 幾啓 岐阜大学

2022.03.14

概要

DNA試料に基づく動物種識別法は、犯罪捜査、野生生物の違法取引調査、法医昆虫学、食品認証などに広く利用されている。法科学分野では、ヒト個人識別について数多くの研究が進められ標準的な解析方法が確立されてきたが、哺乳類や鳥類一般の種識別に関しては標準的な手法が確立されていない。特に、複数種のDNAが混合した劣化試料から低コストで効率的に動物種識別を行う手法の確立が求められている。

論文は4章から構成される。第1章では、本研究の背景と目的をまとめた。第2章では、ミトコンドリアDNA(mtDNA)の分析に基づくこれまでの手法を評価した。第3章では、新しいアッセイ法の開発およびその信頼性に対する詳細な評価を実施した。第4章では、本研究から得られる結論がまとめた。

第2章では、哺乳類の種判別に広く用いられてきたmtDNAに基づく手法を比較検討した。特に、複数種のDNAが混合した試料からの識別という観点で、それぞれの手法の信頼性および汎用性を評価した。現在最も広く用いられているmtDNAシークエンス解析が混合試料の分析においては脆弱であることを示した。その一方、近年技術的進歩が著しい次世代シークエンシング技術を用いたDNAメタバーコーディング解析は、混合試料の分析には強いものの、費用面と時間面で高コストであり、現場の法科学的検査機関が日常的に扱う手法としては適さないことを示した。これらの手法に対し、蛍光マルチプレックスPCR法は低コストで混合試料中の動物種識別能力に優れており、今後も重要な役割を果たしうることを明らかにした。

第3章では、共通プライマーによるmtDNAのD-loop領域の増幅と、種特異プライマーによるcytb領域の増幅とを組み合わせた蛍光マルチプレックスPCRに基づく種識別法を開発した。その結果、日本で日常的に出会う可能性のある動物種をほぼカバーする哺乳類22種および家禽4種の同時識別が可能となった。検出下限がmtDNA100コピーという高い検出感度を有し、混合DNA試料に関しても50:1~1:1までの様々な混合比で高い識別能力を有することを示した。さらに、ScientificWorkingGrouponDNAAnalysisMethods(SWGDAM)のガイドラインに基づいて、手法の信頼性等に関するバリデーションを実施した。

以上、本研究で開発したアッセイ法は低コストである一方で劣化した混合DNA試料にも対応可能であり、識別可能な種数も多い。したがって、日本の法科学分野における標準的な動物種識別法として広く使用されていくことが期待される。

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