长岭发垫刃线虫(Trichotylenchus changlingensis)双重PCR检测方法研究

正长岭发垫刃线虫(Trichotylenchus changlingensis)是一种迁移性植物外寄生线虫,该线虫于2011年首次在我国吉林省长岭县发现,并根据其形态特征及最新分类系统将其归为发垫刃属1,2]。该线虫能引起玉米叶片黄化,植株矮化,茎基部开裂,茎节缩短等症状,该病发生率普遍在21%～67%,给玉米生产造成了严重影响3]。

Development of duplex PCR assay for detection of Trichotylenchus changlingensis

In order to simply, rapidly and accurately identify Trichotylenchus changlingensis, the ITS region of 8 different populations of T. changlingensis was amplified by PCR using a pair of universal primer F194/5368r of nematode. Subsequently, a pair of specific primer, TC-F1/TC-R1, was designed according to the ITS sequence of T. changlingensis and used to establish a duplex PCR system with the combining primer pair D2A/D3B which was a universal primer to detect plant parasitic nematodes and used as internal reference in this study. The results of this study showed that a fragment of 1 057 bp was amplified from 8 T. changlingensis populations using the primer pair F194/5368r, and a specific band of 520 bp was amplified by TC-F1/TC-R1 which displayed a high specificity. Under the duplex PCR system, the anticipated PCR products obtained from T. changlingensis were 520 bp and 780 bp, respectively, and the detection sensitivity was 0.125 ng·μL^-1 of DNA. This system showed the advantages of high sensitivity, strong stability, time-saving and simple operation, and is suitable for the rapid detection of T. changlingensis.