利用环介导等温扩增技术检测黑白轮枝菌

Rapid detection of Verticillium albo-atrum using a loop-mediated isothermal amplification assay

[目的]黑白轮枝菌(Verticillium albo-atrum)能够危害苜蓿等多种作物,是我国重要的检疫性病原真菌。我们基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),建立基于颜色判定的简单、快速和灵敏的黑白轮枝菌检测方法。[方法]基于LAMP技术,通过比对分析黑白轮枝菌与其相似种不同靶标序列间的差异,选取Tub(β-tublin,编码β-微管蛋白)基因作为靶标基因,设计并筛选了4条特异性强、灵敏度高的LAMP引物和1条环引物,在此基础上建立了检测黑白轮枝菌的LAMP体系,并进行特异性、灵敏度试验及田间发病组织的检测。该方法在62℃等温条件下进行核酸扩增反应70 min,扩增前加入染料HNB(羟基萘酚蓝),反应后根据染料颜色变化判定扩增结果。[结果]特异性试验中,仅黑白轮枝菌菌株DNA扩增后呈天蓝色的阳性反应,而在其他真菌的供试菌株中均呈紫色的阴性反应。该方法的最低检测限为1 pg·μL^(-1)。Tub-LAMP技术能够检测出发病苜蓿植物组织中的目标菌,在采自新疆的7份疑似样本中检测到3份阳性。在土壤中人工添加孢子的试验中,Tub-LAMP技术能够从每0.25 g土壤含有10个孢子和每10 g苜蓿种子含有50个孢子中检测出该病菌。[结论]该方法的建立为黑白轮枝菌的检疫及其所致病害的诊断提供了新的技术。

[Objectives] Verticillium albo-atrum is an important quarantine fungal pathogen in China,which can infect alfalfa and other crops. Based on loop-mediated isothermal amplification technology （LAMP）,we developed a simple,rapid and sensitive method for the detection of V.albo-atrum.[Methods] By comparing different sequences of V.albo-atrum with those of relative species,we selected Tub gene（encoding β-tubulin）as the target gene,designed and selected four specific and sensitive LAMP primers and a loop primer to detect V.albo-atrum. The LAMP assay efficiently amplified the target gene in 70 min at 62℃ and was evaluated for specificity,sensitivity and on diseased alfalfa tissues. The result can be directly visualized to naked eyes by adding HNB （hydroxynaphthol blue）prior to the amplification.[Results] The specificity was evaluated against V.dahliae and other fungal isolates. A positive color（sky blue）was only observed in the presence of V.albo-atrum whereas all other isolates showed purple color. The detection limit of the LAMP assay for V.albo-atrum was 1 pg·μL^-1 and its detection sensitivity for soil samples and seeds were respectively 10 conidiospores per 0.25 g soil and 50 conidiospores per 10 g seeds. V.albo-atrum was detected from 3 among 7 suspected diseased-alfalfa tissues collected from Xinjiang.[Conclusions] This method provides a new technology for the diagnosis and quarantine of V.albo-atrum.