松材线虫SCAR标记与系列分子检测技术及试剂盒研制
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本课题通过松材线虫与拟松材线虫、其它线虫之间的遗传差异，筛选出了松材线虫、拟松材线虫的特异RAPD片段、SCAR标记片段和相应的随机引物、SCAR特异引物，标记出了松材线虫非放射性杂交探针，设计、合成了松材线虫实时定量PCR的特异引物与探针，成功构建了松材线虫SCAR标记检测试剂盒、实时定量PCR检测试剂盒以及杂交检测试剂盒，开发出了一系列实用的松材线虫分子快速检测和鉴定技术与方法。 本课题首次成功地从线虫遗传物质入手，为松材线虫检疫鉴定建立了一套准确有效的分子标记系统，克服了传统的形态鉴定需时长、鉴定难度大、不能对幼虫和雄虫进行鉴定等的不足，实现了对松材线虫幼虫、雌雄成虫均可进行检测鉴定的目标，为松材线虫病新疫区疫点的确定和防止疫木的传播提供了快速、准确和安全的松材线虫鉴定和检测方法，具有很强的实用性。其检测手段先进，数据翔实可靠，检测结果易于判读，准确性达100%。鉴定委员会认为，该项目技术先进，创新性强，总体技术水平达到同类研究的国际先进水平。
This project screened out species-specific RAPD fragments, SCAR marker fragments, corresponding random primers and SCAR-specific primers for *Bursaphelenchus xylophilus* (pine wood nematode, PWN) and *Bursaphelenchus mucronatus* based on the genetic differences between PWN, *B. mucronatus* and other nematode species. It also developed non-radioactive hybridization probes for PWN, designed and synthesized specific primers and probes for real-time quantitative PCR detection of PWN, successfully constructed SCAR marker detection kits, real-time quantitative PCR detection kits and hybridization detection kits for PWN, and developed a series of practical rapid molecular detection and identification techniques and methods for PWN. This project, for the first time, successfully established an accurate and effective molecular marker system for the quarantine identification of PWN starting from the genetic material of nematodes, overcoming the shortcomings of traditional morphological identification, such as long time consumption, high identification difficulty, and inability to identify larvae and males. It achieved the goal of detecting and identifying all developmental stages of PWN, including larvae, females and males, providing rapid, accurate and safe identification and detection methods for determining new epidemic foci of pine wilt disease and preventing the spread of infested wood, with strong practicality. The detection method is advanced, with comprehensive and reliable data, easy-to-interpret results, and 100% accuracy. The appraisal committee concluded that this project features advanced technology and strong innovation, with its overall technical level reaching the international advanced level in the same research field.
