樹木褐根病的恆溫環狀擴增法（LAMP）檢測方法之研發

Development of the Detection Method for Tree brown Root Rot Disease Based on the Loop-mediated Isothermal Amplification (LAMP)

黃裕星(Y. H. Huang) ； 陳昭翰(C. H. Chen) ； 洪挺軒(T. H. Hung) ； 吳孟玲(M. L. Wu) ； 張東柱(T. T. Chang)

《植物病理學會刊》 23卷1期 (2014/03) Pp. 1-13

https://doi.org/10.6649/PPB.201403_23(1).0001

摘要

褐根病（brown root rot disease）是國內林木目前最嚴重的病害，是由病原真菌Phellinus noxius所引起。由於樹木罹病初期不易以外部病徵來診斷，為掌握防治先機，本研究應用恆溫環狀擴增法（LAMP, Loop-mediated isothermal amplification）開發新的快速病害檢測技術，以期提高褐根病診斷效率。培養的褐根病菌絲以一般法與簡易快速法分別抽取DNA，發現以一般法為佳，簡易快速法所得DNA則雜質較多；另以傳統抽取法、改良抽取法及商業化套組抽取法分別去純化感染褐根病的樹木樣本DNA，結果篩選出改良抽取法做為標準的DNA純化方法。本研究設計三個LAMP引子組（命名為Phenox 1、2與3）針對Phellinus gilvus、P. igniarius、P. linteus、P. inermis、P. pachyphloeus、P. pectinatus、P. prunicola及P. torulosus及P. noxius等菌種進行PCR增幅反應測試，偵測結果顯示第一引子組（Phenox 1）對P. noxius 有專一性的增幅反應，對於其餘八種Phellinus spp.則無反應；而第二與第三引子組（Phenox 2與Phenox 3）除了對於P. noxius之核酸進行增幅反應外，還分別會對於P. igniarius，P. pectinatus出現增幅反應。本論文所研發的改良式褐根病樹木樣本DNA抽取法搭配所設計的Phenox 1引子組，可對P. noxius進行LAMP專一性增幅，靈敏度較先前研發的PCR檢測法可提升10倍，可做為另一種褐根病快速分子診斷法。

關鍵字

褐根病 ； 恆溫環形核酸增幅法（LAMP） ； 檢測

並列摘要

Brown root rot caused by the pathogenic fungi, Phellinus noxius, is the most serious disease of domestic trees. It is not easily diagnosed by its external symptoms especially in the early stage of infection. To elevate the diagnostic accuracy and efficiency, a new method based on the loop-mediated isothermal amplification (LAMP) was developed for the molecular detection of Phellinus noxius in this study. The fungal DNA was extracted from the mycelia cultured on media with general and simple extraction methods, and the results showed that the general method obtained purer DNA than the simple method. The DNA was extracted from the tree tissues infected by Phellinus noxius with 3 different extraction methods such as the traditional method, improved method and commercial kit respectively, and the comparative results demonstrated that the improved method was better and selected to be the standard protocol used in the LAMP detection for Phellinus noxius. Totally 3 primer sets (named Phenox 1, 2 and 3) were designed for the LAMP assay of Phellinus noxius. Our data reported that the Phenox 1 primer set showed positive for Phellinus noxius but negative for Phellinus gilvus, P. igniarius, P. linteus, P. inermis, P. pachyphloeus, P. pectinatus, P. prunicola and P. torulosus in the LAMP assays. The Phenox 2 and 3 primer sets showed positive for P. noxius, P. igniarius and P. pectinatus whereas they showed negative for Phellinus gilvus, P. linteus, P. inermis, P. pachyphloeus, P. prunicola and P. torulosus. Our devised LAMP detection of Phellinus noxius with Phenox 1 primer set and the improved extraction method from the diseased tree tissues is more sensitive (10 folds) than the previously developed PCR method, and it may be another rapid molecular diagnosis for the brown root rot disease.

並列關鍵字

brown root rot disease ； Phellinus noxius ； loop mediated isothermal amplification (LAMP)