Development of two alternative Loop-mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

作者：Kang J S; Kim A Y; Han H R; Moon Y S; Koh Y H^*

来源：Forest Pathology, 2015, 45(2): 127-133.

DOI：10.1111/efp.12147

Loop-mediated isothermal amplification (LAMP) detection tools have great potential for diagnosing the causal agents of infectious diseases in clinics and in agriculture. In this work, we developed two alternative LAMP protocols for detecting the pathogenic nematode Bursaphelenchus xylophilus, causal agent of pine wilt disease. We first identified a pectate lyase 3 gene as a biomarker for developing a LAMP Detection Kit, as there was no homologue in non-pathogenic nematodes that live in pine timber or bark and show structural similarities to B.xylophilus. The first LAMP protocol used the Genie II equipment and an isothermal master mix containing Geobacillus sp. M 2.0 large fragment DNA polymerase showed approximately 10 times greater sensitivity with a shorter incubation period compared with that of the second LAMP protocol, which utilized a fluorescence metal indicator, calcein and an engineered Bacillus stearothermophilus DNA polymerase I, large fragment (Bst 2.0 DNA polymerase). However, the LAMP reactions with calcein and Bst 2.0 polymerase were the cost-effective method because the reaction could be performed using a simple isothermal block and relatively inexpensive calcein as a fluorescence indicator visible to the naked eye.

• 出版日期2015-4