寡核苷酸管芯片技术检测和鉴别我国不同组植原体

目的]不同组植原体检测和鉴别的特异性探针已有报道,为了筛选出适合于我国不同组植原体检测和鉴别的特异性探针,建立管芯片检测和鉴别植原体技术,并对我国发生的疑似植原体病害进行鉴别。方法]通过PCR扩增结合管芯片杂交技术,对收集到的15种植原体侵染的植物样品及其健康对照进行检测和鉴别。结果]建立了管芯片检测和鉴别植原体技术体系。15种病害样品中,13种获得显著的阳性杂交信号,并且所有的健康对照都呈现为阴性。13种植原体病害依16Sr DNA直接测序可分为16SrⅠ、Ⅱ、Ⅴ、XIX四组植原体。在所有探针中,植原体的通用探针(Pp-502)可以检测到所有确定的植原体样品。16SrⅠ组特异性探针(PpⅠ-465)可以确定16SrⅠ组的泡桐丛枝、苦楝丛枝、桑树萎缩和莴苣黄化4种植原体样品。16Sr II组特异性探针(PpⅡ-629)仅可以确定16Sr II组的花生丛枝、甘薯丛枝和臭矢菜丛枝3种植原体样品。但16Sr V组的枣疯病、樱桃致死黄化和重阳木丛枝及16Sr XIX组的板栗黄化皱缩植原体与其他组专化性探针皆有明显的交叉杂交信号。相比于PCR扩增的凝胶电泳检测,管芯片检测的灵敏度提高了1 000倍。对疑似植原体病害的诊断结果显示河南濮阳的红花槐丛枝的病原应为16Sr V组植原体,福建福州的长春花黄化丛枝应为16SrⅠ组植原体;而北京戒台寺牡丹黄化皱叶和内蒙古包头柳树丛枝未出现任何植原体专化的杂交信号。结论]管芯片杂交技术作为一种检测和鉴别植原体的方法,可应用于我国植原体病害调查和诊断,并为植原体的鉴别和分类提供可靠的依据。

Diagnostics and Detection of Different Groups Phytoplasmas in China Using an Oligonucleotide Microarray on the Platform of ArrayTube

Objective] d the optimal specific probe and develop the detection technique using oligonucleotide microarray on the platform of ArrayTube to detect and identify the phytoplasmas associated with plant disease in China.Method] PCR amplification and microarray hybridization were used to detect 15 symptomatic plants probably infected with phytoplasma and asymptomatic plants as healthy controls collected from different regions in China.Result] Phytoplasma 16S rDNA were detected in 13 of 15 symptomatic plants but not in all the healthy controls. Thirteen phytoplasmas were classified into 16Sr I, 16Sr II, 16Sr V and 16Sr XIX groups. Among 17 tested probes, the universal probe designated Pp-502 could be used to detect all phytoplasmas associated with plant disease. The specific probe designated Pp I-465 for 16Sr I group could be used to detect four phytoplasma strains of 16Sr I group associated with paulownia witches'' broom, chinaberry witches'' broom, mulberry dwarf and lettuce yellows. The probe Pp II-629 for 16Sr II could be used to detect three phytoplasma strains 16Sr II group associated with peanut witches'' broom, sweet potato witches'' broom and cleome witches'' broom. Three phytoplasma strains of 16Sr V associated with jujube witches'' broom, cherry lethal yellows and Bischofia polycarpa witches broom and chestnut yellows crinkle phytoplasma of 16Sr XIX could also be detected by specific probes, but they showed obvious cross hybridization with other group probes. Compared with PCR amplification, the sensitivity of microarray to detect phytoplasma in plant increased by 1000-fold. Phytoplasmas of 16SrI and 16SrV group respectively were detected in periwinkle with symptoms of phyllody and witches'' broom collected from Fujian province and Robinia hispida with symptom of witches'' broom collected from Henan province. While no phytoplasma was detected in peony with symptom of yellowing collected from Beijing and willow with symptom of witches'' broom collected from Inner Mongolia Autonomous Region.Conclusion] The oligonucleotide microarray on the platform of ArrayTube could be used as a method to investigate phytoplasmas in China, and could provide a sound basis for the phytoplasma identification and classification.