恆溫圈環形核酸增幅偵測技術於水稻徒長病菌篩檢之適用性探討

The feasibility of loop-mediated isothermal amplification (LAMP) detection technique for rice bakanae disease screening application plants

"水稻徒長病為我國水稻重要病害，其病原真菌存於穀粒中，於育苗時期感染水稻苗，發病嚴重時對稻穀產量及品質影響甚鉅。本研究針對此病害於種苗時期之篩選需求，建立恆溫圈環形核酸增幅法（Loop-mediated isothermal amplification, LAMP）快速檢測技術。該技術挑選水稻徒長病菌cps/ks基因，設計專一性LAMP引子。透過增幅效率結果分析，最終挑選在最適反應溫度63°C條件下表現最佳之引子組CPS-ID4。當目標核酸濃度超過50 pg/μl時，可在90分鐘內被該引子組成功偵測。本研究所開發之技術專一性遠高於傳統PCR方法，可排除同屬中其他8種近似鎌孢菌之偵測，且能有效檢出14株不同來源之徒長病菌分離株。挑選9個水稻品種稻苗進行應用測試，發現該技術可成功偵測人工接種感染但尚未發病之全部受試品種之7天大水稻苗。本研究中開發之LAMP引子組，搭配hydroxy naphthol blue（HNB）染劑和偵測試紙，能於偵測終端快速顯示偵測結果，有效減少完成檢測判讀所需時間，亦大幅提升本技術應用於水稻苗期檢測和植物檢疫的潛力。"

"Bakanae disease is an important rice disease in Taiwan. The pathogen remains in the grains and infects the rice seedling during the nursery stage, which severely affects the yield and the quality of grains when the disease outbreaks. Owing to the needs of screening out the disease-infected rice seedlings, a Loop-mediated isothermal amplification (LAMP) rapid detection technique was developed. Specific LAMP primer sets were designed to target the cps/ks gene of the pathogen Fusarium fujikuroi. After the amplification efficiency analysis of the optimum reaction temperature at 63°C, the best performed primer set, CPS-ID4, was finally picked. When the target pathogen gDNA concentration was higher than 50 pg/μl, it could be detected by CPS-ID4 in less than 90 minutes. In addition, the specificity of this assay was much higher than the traditional PCR method. It is capable of excluding 8 other Fusarium species that are closely related to F. fujikuroi while being able to detect all 14 tested F. fujikuroi isolates. Nine rice cultivar seedlings were selected for F. fujikuroi inoculation to evaluate the assay's application potential. The positive outcomes demonstrated the successful detection ability of this assay on the symptomless 7-day-old rice seedlings. Lastly, this study showed the CPS-ID4 LAMP primer set worked well with rapid analysis techniques including hydroxy naphthol blue (HNB) dye and lateral flow strip, and that hugely improved the application potential of this assay for rice bakanae disease detection in the seedling nursery and plant quarantine."