Citation

Miyazaki H, Taniguchi M. Shokuhin Eiseigaku Zasshi 2024; 65(3): 53-60.

Vernacular Title

リアルタイムPCRを用いた有毒植物の属鑑別法の開発

Copyright

(Copyright © 2024, Food Hygienic Society of Japan)

DOI

10.3358/shokueishi.65.53

PMID

39034136

Abstract

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan(®) probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

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有毒植物の属を迅速に鑑別する方法を開発した．検知手法はTaqMan® プローブ法を用いたリアルタイムPCRとし，増幅領域は葉緑体DNAの"trnL（UAA）-intron"領域および"trnL-trnF intergenic spacer"領域とした．検知対象は，我が国で食中毒事例が多数報告されている6属（トリカブト，イヌサフラン，シュロソウ，キダチチョウセンアサガオ，ハシリドコロ，スイセン）とした． DNAの抽出には30分程度で完了する組織溶解液を，リアルタイムPCR試薬はこの組織溶解液に対応したマスターミックスを使用した．これらにより，DNA抽出から属特定までの全工程を4～5時間で完了可能であった．検知感度は6属すべての植物においてDNA量として1 pgであり，粗抽出DNA溶液でも増幅曲線が得られた．模擬調理（茹でる）を行った3属の植物試料においても増幅曲線が得られた．本法により迅速に6属の有毒植物を検知できる可能性が示唆された．

Language: ja

Keywords

Time Factors; *Plants, Toxic/classification/genetics; *Real-Time Polymerase Chain Reaction/methods; Aconitum/genetics/classification/chemistry; chloroplast DNA; DNA, Chloroplast/genetics/analysis; DNA, Plant/genetics/analysis; food poisoning; Foodborne Diseases/prevention & control; PCR real-time PCR; poisonous plant; Sensitivity and Specificity; Veratrum/genetics/chemistry/classification