松材线虫两种实用分子检测技术

为检验已开发的SCAR标记与实时PCR两种分子检测方法鉴定松材线虫的可靠性，本文选用线虫未知种样品7个以及已知种样品3个为实验材料，采用上述两种分子检测方法进行检测，并且对7个线虫未知种样品进行形态鉴定。结果表明：1）运用SCAR标记检测，第2、3、4、7号4个未知种样品与8号松材线虫样品出现了一条清晰、明亮的860b...

Two kinds of applied molecular skills to detect Bursaphelenchus xylophilus

In order to determine the reliability of two methods, SCAR marker and real-time PCR, developed for detecting pine wood nematode, seven pine wood isolates and three identified nematode strains (Bursaphelenchus xylophilus, B. mucronatus and Aphelenchoides macronucleatus) were assayed with the two methods. The traditional method of morphological identification was also used to determine the seven isolates samples. Results showed that 1) with the method of SCAR marker, isolates 2, 3, 4 and 7 showed a common specific electrophoretic band of 860 bp, as did by pine wood nematode B. xylophilus, whereas isolates 1, 5 and 6, and B. mucronatus and Aphelenchoides macronucleatus did not produce the specific band. That suggests that isolates 2, 3, 4 and 7 contained B. xylophilus, while isolates 1, 5 and 6 did not. 2) With the method of real-time PCR identification, fluorescent signal was detected from samples prepared from isolate 2, 3, 4, 7 and strain B. xylophilus, on the contrary, the rest isolates showed no signals and cycle threshold (Ct values). 3) With morphological identification, it was revealed that isolate 2, 4 and 7 contained B. xylophilus while isolate 3 contained B. xylophilus as well as other nematodes. However, isolate 1, 5 and 6 did not contain B. xylophilus. Therefore, the two molecular methods showed a consistent result with morphological identification. Moreover, the two methods are highly specific and time-saving. SCAR marker method costs two hours to perform, while real-time PCR method takes only one hour. Both techniques make it possible to determine rapidly existence of juvenile pine wood nematodes with a simple and easily readable result.