PCR偵測法應用在台灣二葉松萎凋病病原研究

PCR Detection Applied in a Study of the Causal Agent of Pine Wilt Disease in Taiwanese Red Pine, Pinus taiwanensis

吳孟玲(Meng-Ling Wu) ； 張東柱(Tun-Tschu Chang) ； 傅春旭(Chuen-Hsu Fu) ； 馮雅智(Ya-Chin Feng) ； 王涼綢(Laing-Chour Wang)

《台灣林業科學》 19卷1期 (2004/03) Pp. 89-96

https://doi.org/10.7075/TJFS.200403.0089

摘要

本研究開發簡易松材線蟲(Bursaphelenchus xylophilus)的核酸抽取法，線蟲全核酸抽取僅需2h完成；同時建立高敏感度、快速及正確偵測松材線蟲的PCR技術，利用松材線蟲衛星核酸(satellite DNA monomer)基因序列，設計對松材線蟲專一性的引子對，藉聚合酵素連鎖反應（polymerose chain reaction，簡稱PCR）正確區分松材線蟲與擬松材線蟲(B. mucronatus)，在黏合溫度為54℃之PCR反應下對松材線蟲之全DNA可增幅出單一條大小為131 bp之PCR產物。針對分離自台灣二葉松北部及中部地區二葉松之線蟲分離株，進行PCR偵測結果顥示造成台灣二葉松萎凋病之病原線蟲，應是屬於松材線蟲。PCR偵測及點漬雜配法(dot hybridization，DH)皆可對國內外松材線蟲株獲得正反應，對擬松材線蟲株則呈負反應。核酸序列分析顯示台灣二葉松松材線蟲分離株與日本分離株有94%相似度，且同樣為A+T-rich。

關鍵字

PCR偵測法 ； 台灣二葉松 ； 松材線蟲

並列摘要

Based on the published sequences of satellite DNA monomer, of B. xylophilus and B. mucronatus, we designed specific primers to detect B. xylophilus by PCR detection. This PCR-based assay is a quick method for correct differentiation between B. xylophilus and B. mucronatus, which can be accomplished within 5.5 h including a 2-h DNA extraction from nematodes, 3-h PCR thermocycles, and a 0.5-h electrophoretic analysis. A specific band with a size of 131 bp could be amplified from the DNA extract of B. xylophilus, whereas no product was amplified from that of B. mucronatus. Using this method, we demonstrate that the newly discovered nematode from Taiwanese red pine belongs to a pathotype of B. xylophilus. This PCR-based assay was able to detect all samples of B. xylophilus collected from foreign countries; however, all foreign samples of B. mucronatus had negative results for the detection. Dot hybridization with DNA probes specific to B. xylophilus obtained the same results as the PCR assay. DNA sequence analysis revealed that the amplified DNA fragments of 131 bp of Taiwanese and Japanese isolates of B. xylophilus shared 94% homology.

並列關鍵字

polymerase chain reaction (PCR) ； Taiwanese red pine ； pinewood nematode (Bursaphelenchus xylophilus)