大蕉枯萎病菌二重PCR分子快速检测技术的建立

Development of duplex PCR molecular rapid detection for fusarium wilt of Dajiao

【目的】基于大蕉枯萎病菌2个不同分化谱系特有的基因序列,建立大蕉枯萎病菌的二重PCR分子快速检测技术,为该病害预测及有效防控提供理论依据。【方法】基于大蕉枯萎病菌特有的rDNA基因间隔区(IGS)序列设计得到多对PCR引物,分别以广东3个粉蕉枯萎病菌株和8个大蕉枯萎病菌株、2个广西和海南的粉蕉枯萎病菌株、5个其他尖孢镰孢菌菌株和5个外围菌株的基因组DNA为模板进行PCR扩增,基于扩增结果从中筛选出大蕉枯萎病菌特异性引物,对其特异性和灵敏度进行检测,并确定二重PCR检测体系的含菌量检测下限。【结果】基于大蕉枯萎病菌特有的基因序列设计得到多对PCR引物,经筛选验证获得2对可用于检测大蕉枯萎病菌2个不同谱系(LineageⅠ和Ⅱ)的特异性引物,可直接应用于大蕉种植土壤和植株中的大蕉枯萎病菌检测,扩增片段分别为755和590 bp。当扩增条带为755 bp时,表明样本中含有大蕉枯萎病菌;当扩增条带为590 bp时,表明样本中含有粉蕉或大蕉枯萎病菌。建立的二重PCR检测体系检测大蕉枯萎病菌菌丝的灵敏度可达0.4μg,检测土壤的含菌量下限为每克土壤40个病原菌孢子。【结论】建立的二重PCR检测技术实现一次PCR反应同时检测并区分大蕉枯萎病菌2个谱系及广东地区的粉蕉枯萎病菌优势菌群,可用于无病大蕉和粉蕉种苗及种植土壤的检测与筛选。

【Objective】The objective of this study was to establish duplex PCR molecular detection for fusarium wilt strain of Dajiao based on gene sequences specific to 2 different differentiation lineages of Dajiao fusarium wilt strain,which could provide theoretical basis for prevention and control of banana fusarium wilt.【Method】Multiple pairs of PCR primers were designed based on rDNA intergene region(IGS)unique to pathogen of fusariun wilt pathogen of Dajiao,DNAs of 8 fusariun wilt strains of Dajiao,3 fusarium wilt strains of Fenjiao from Guangdong,2 fusarium wilt strains of Fenjiao from Guangxi and Hainan respectively,5 F.oxysporum strains and 5 outgrouped strains were amplified by PCR as templates to screen the specific primers for the fusarium wilt strain of Dajiao.The specificity and sensitivity of the primers were tested,and the lower limit of strain content detection of duplex PCR system was determined.【Result】Several pairs of PCR primers were designed based on the specific gene sequence of fusarium wilt strain of Dajiao.After screening and verification,2 pairs of specific primers were obtained that could be used to detect 2 different lineages of fusarium wilt strains(LineageⅠand LineageⅡ),which could be directly applied to the detection of Dajiao fusarium wilt strains in Dajiao planting soil and Dajiao plants.The amplified fragments were 755 and 590 bp,respectively.When the amplified band was 755 bp,it indicated that Dajiao fusarium wilt strain was contained in the sample.When the amplified band was 590 bp,it indicated that the sample contained the strains of Fenjiao or Dajiao fusarium wilt.The sensitivity of the duplex PCR detection system for Dajiao fusarium wilt strain hypha was up to 0.4μg,and the lower limit of soil strain content was 40 spores per gram of soil.【Conclusion】The established duplex PCR detection technique can simultaneously detect and distinguish two lineages of fusariun wilt strains of Dajiao and the dominant group of fusarium wilt of Fenjiao in Guangdong area in one PCR reaction,which can be used for the detection and screening of disease-free Fenjiao and Dajiao seedlings and planting soil.