一种适于PCR和LAMP检测的松木中松材线虫DNA快速提取方法

摘要

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 OD260 / 280 of the three methods was in a descending order of CTAB method > Proteinase K ≥Chelex-100 method. The OD260 / 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 OD260 / 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.%【目的】本研究旨在探索快速、简便地直接从病木中提取松材线虫 DNA 的方法。【方法】运用 Chelex-100结合异硫氰酸胍蛋白变性缓冲液建立新的高效快速提取微量木块中线虫 DNA 的方法，并通过普通 PCR 与环介导等温扩增( LAMP)对提取的 DNA进行检测验证。【结果】建立了提取线虫 DNA 的新方法 Chelex-100法，并对影响提取效率的Chelex-100浓度、冻融时间和煮沸时间进行了优化。Chelex-100法最适Chelex-100终浓度为1.5%( W/V)，最适冻融时间为5 min，最适煮沸时间为8 min。与传统的 CTAB法和蛋白酶 K 法比较，Chelex-100法提取的 DNA得率高，相同条件下，该方法提取的 DNA浓度远远大于常规方法。3种方法的 OD260/280比值从大到小依次为CTAB法＞蛋白酶K法≥Chelex-100法，Chelex-100法的OD260/280值显著低于CTAB法，而略低于或等同于蛋白酶K法，但这并不影响对提取的 DNA 进行 PCR 扩增。采用 Chelex-100法提取松木中松材线虫的 DNA，结合常规的PCR和 LAMP检测，检测灵敏度高，特异性强，稳定性好；提取的松材线虫 DNA 的75倍稀释液再稀释32倍后进行PCR扩增，扩增产物电泳依然有清晰的条带。用新方法提取病木中线虫的 DNA 后进行检测，所有带病松木样品的检测结果均呈阳性，而健康黑松、马尾松、油松木屑及盘多毛孢样品的检测结果均呈阴性。此外，该提取方法简便快速，20 min内即可完成整个 DNA的提取；经济方便，试剂保藏无特殊要求，单个样品提取耗费低于3.5元。【结论】Chelex-100法是一种快速有效的提取松木中松材线虫DNA的方法，此方法结合PCR和LAMP检测技术将进一步提高松材线虫的检测效率、灵敏度和准确性，可为松材线虫的野外检测提供可靠的技术依据。