两种分子技术检测松木中松材线虫的效果评价
Evaluation of two molecular methods for detection of pine wood nematode Bursaphelenchus xylophilus in pine woods

为更好地解决当前松材线虫Bursaphelenchus xylophilus检测工作中频现的假阴性问题，通过不同分子技术检测效果的比较评价，摸索出了一套可提高松材线虫检测准确性的技术体系。针对早期感病松木的低线虫量，建立了线虫的最大量分离方法和基于rDNA ITS序列的分子检测体系，该体系可高效检测出单条松材线虫，准确率达93.75%。以来自浙江省不同疫区的96份松木样品为检测材料，比较了SCAR标记和ITS序列2种分子检测技术的阳性率。结果显示，通过首次PCR，ITSⅠ和ITSⅡ序列的检测阳性率分别为52.08%和55.21%，SCAR标记的检测阳性率为30.21%；通过第二次或巢式PCR，ITSⅠ和ITSⅡ序列的检测阳性率提高到了97.92%和100.00%，SCAR标记的检测阳性率提高到了59.38%，表明基于ITS序列的检测阳性率明显高于SCAR标记，且通过第二次或巢式PCR方法可进一步提高检测灵敏度，降低假阴性率。因此，通过线虫的最大量分离并基于rDNAITS序列的分子检测可明显提高检测准确性，更适用于松材线虫的常规检测。
The false negative problem occurs frequently in detecting pine wood nematode Bursaphelenchus xylophilus. To explore a new technology which can significantly improve the detection accuracy, the detection capabilities of two molecular techniques were compared and evaluated. For a small number of B. xylophilus from the early disease-infected pine trees, the new technology established in this study was highly efficient, and could even detect single nematode with a detection accuracy of 93.75%. In addition, 96 pine samples from different epidemic areas of Zhejiang Province in China were collected and the detection capabilities of two methods using SCAR markers and rDNA ITS were compared. The results showed that the positive rates of ITSⅠ and ITSⅡ were 52.08% and 55.21%, respectively, which were higher than those by using SCAR markers (30.21%). The detection sensitivity could be further increased by using two round PCR/nested PCR, in which the positive rates of ITSⅠ and ITSⅡ were 97.92% and 100.00%, respectively, higher than those with SCAR markers (59.38%), and the probability of false negative was reduced significantly. The results indicated that the molecular detection technology based on ITS could improve the detection accuracy significantly, and was more suitable for routine detection of B. xylophilus.