松材线虫实时PCR检测技术

Detection Technique of Bursaphelenchus xylophilus Using Real Time PCR

根据松材线虫核糖体DNA内部转录区设计了一对特异引物F11/R11与探针TaqMan-11,构成松材线虫实时PCR检测方法。采用该检测方法对分离自枯死松树体内的松材线虫、拟松材线虫、大核滑刃线虫、霍夫曼尼伞滑刃线虫和长尾属线虫进行检测。结果显示,所有松材线虫株系都能够检测到荧光信号,而拟松材线虫、大核滑刃线虫、霍夫曼尼伞滑刃线虫、长尾属线虫以及对照均没有检测到荧光信号。表明探针Taq-Man-11与引物组合对松材线虫具有很强的特异性。此外,利用检测方法,在10μL的反应体系中,最少可以检测到0.005pg的松材线虫DNA含量。实时PCR也可以成功地检测出单条松材线虫。

In this paper, a set of primers （F11/R11） and probe （TaqMan-11） specific for Bursaphelenchus xylophilus was designed to target the ribosomal DNA internal transcribed spacer（ITS） region, which composed the real-time polymerase chain reaction （PCR） assay. Then, B. xylophilus, B. rnucronatus, B. hofmanni, A. macronucleatus and Seinura sp. separated from dead pine were detected a using this real-time PCR assay. The results showed that fluorescent signals were detected for all B. xylophilus isolates and there was no signal for B. rnucronatus, B. hofmanni, Aphelenchoides macronucleatus, Seinura sp. and water （control）. This indicated that the probe （TaqMan-11）and primers （F11/R11） were highly specific for B. xylophulus. Besides, the assay was very sensitive, detecting as little as 0. 005 pg of B. xylophilus DNA in 10μL volume. The real-time PCR assay also successfully detected single pinewood nematodes, and it should be very useful for quarantine purposes.