关键词: 松材线虫, DNA提取, Chelex-100

Abstract: [Objective] It is crucial for detection of the pine wood nematode (PWN) to develop a rapid and economic method of DNA extraction from the infested wood samples by Bursaphelenchus xylophilus. This study aims at developing a method for rapidly extracting DNA of Bursaphelenchus xylophilus from the infested pine wood.[Method] In this study, a new method was developed to directly extract the nematode DNA from the infested wood chips by using Chelex-100 combined with alkaline guanidine thiocyanate. The extracted DNA was amplified by polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). [Result] The Chelex-100 was used to extract the nematode DNA and the factors, including Chelex-100 concentration, freezing and boiling time, affecting the extraction efficiency were optimized. The result showed that the optimum Chelex-100 final concentration was 1.5% (W/V), the optimum thawing time was 5 min, and the optimum boiling time was 8 min. The DNA concentration extracted by the Chelex-100 method was significantly higher than that extracted by CTAB and Proteinase K method under the same condition. The ratios of OD_260/280 of the three methods was in a descending order of CTAB method > Proteinase K ≥Chelex-100 method. The OD_260/280 value of extraction with Chelex-100 method was significantly lower than that with the CTAB method, but slightly lower than, or equal to that with the Proteinase K method, however the low OD_260/280 value did not affect the extracted DNA used for PCR amplification. The electrophoresis strip of the specific PCR amplification of the 75×32 times dilution of the nematode DNA extracted by Chelex-100 method was still clear. The results of PCR and LAMP indicated that the detection of PWN using the Chelex-100-extracted-DNA was sensitive, specific, and stable. With the method the entire infested wood samples by PWN were detected positive, however, the healthy pine samples, including Pinus thunbergii, P. massoniana, P. tabuliformis and Pestalotiopsis microspore, were detected negative. Furthermore, the new method is simple and rapid, with which only 20 minutes are needed without any other special requirements, and the cost was only 3.5 yuan per sample.[Conclusion] The Chelex-100 method is a rapid and valuable method for DNA extraction from PWN-infested wood samples, it can further improve the efficiency, sensitivity and accuracy of the PWN detection together with PCR and LAMP techniques for PWN detection under field conditions.

Key words: Bursaphelenchus xylophilus, DNA extraction, Chelex-100