基于mtCOI基因的三种短体线虫多重PCR检测  

Multiplex-PCR Detection of Three Pratylenchus Species Based on mtCOI Gene

作　　者：秦鑫 王亚东 李红梅 于家荣 王暄 QIN Xin;WANG Ya-Dong;LI Hong-Mei;YU Jia-Rong;WANG Xuan(College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests,Ministry of Education,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室,南京210095

出　　处：《农业生物技术学报》2020年第3期562-570,共9页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项(201503114);国家自然科学基金(31471751;31872923)。

摘　　要：短体线虫(Pratylenchus spp.)是严重破坏世界农业生产的一类植物寄生线虫,建立针对该类线虫的快速检测方法,对根腐线虫病的综合防控至关重要。本研究基于线粒体细胞色素氧化酶亚基Ⅰ(mitochondrial cytochrome oxidaseⅠ, mtCOI)基因序列,分别设计了咖啡短体线虫(P. coffeae)、落选短体线虫(P. neglectus)和斯克里布纳短体线虫(P. scribneri)的特异性上游引物Pc-m193-F、Pn-m280-F和Ps-m66-F以及三者通用的下游引物Pcns-m-R,扩增产物预期大小分别为225、137和351 bp,检测灵敏度分别为1/1000、1/10 000和1/100单条成虫;经过不同引物配比及退火温度等反应条件优化,当引物Pc-m193-F、Pnm280-F、Ps-m66-F、Pcns-m-R浓度分别为0.4、0.4、0.8、1.2μmol/L,退火温度为54℃时,上述引物组合在1个PCR反应体系中能够同时检测出3种靶标线虫。运用该多重PCR体系对18个短体线虫样品进行检测,结果与已知鉴定结果相一致。本研究建立的多重PCR体系可快速检测咖啡短体线虫、落选短体线虫和斯克里布纳短体线虫单一或复合侵染样品,可为我国小麦(Triticum aestivum)根腐线虫病的诊断与综合治理提供技术支持。The Pratylenchus species are important plant-parasitic nematodes and seriously damage the agricultural production worldwide. The establishment of a rapid method for detecting Pratylenchus species is significant to the integrated management of root-lesion nematode diseases. Based on the comparisons of mitochondrial cytochrome oxidase Ⅰ(mtCOI) gene sequences from Pratylenchus species, primers were designed, including 3 forward primers Pc-m193-F, Pn-m280-F and Ps-m66-F specific to P. coffeae, P.neglectus and P. scribneri, respectively, and one common reverse primer Pcns-m-R. The PCR products were225, 137 and 351 bp, respectively, and the PCR sensitivities were 1/1 000, 1/10 000 and 1/100 of individual adult nematode, respectively. The multiplex-PCR system was established successfully after optimizing the concentration ratio of different primers and the annealing temperature. Three Pratylenchus species can be simultaneously and specifically detected using the multiplex-PCR by the combination of primer Pc-m193-F,Pn-m280-F, Ps-m66-F and Pcns-m-R with concentration of 0.4, 0.4, 0.8 and 1.2 μmol/L, respectively, and the annealing temperature with 54 ℃. The detection of 18 Pratylenchus samples using the multiplex-PCR was carried out, and the results were consistent with previous identification. The established multiplex-PCR system could be used to quickly detect the P. coffeae, P. neglectus and P. scribneri either in the samples of single or mixed species infection. This technique could provide the support for diagnosis and integrated management of Triticum aestivum root-lesion nematode diseases in China.

关 键 词：多重PCR 线粒体细胞色素氧化酶亚基Ⅰ基因(mtCOI) 短体线虫 特异性 

分 类 号：S432.45[农业科学—植物病理学]