中国马铃薯疮痂病菌快速PCR检测技术  

Rapid PCR Detection Technology of Common Scab Pathogens in China

作　　者：郭凤柳[1] 张海颖[1] 于秀梅[1] 赵伟全[1] 刘大群[1] 

机构地区：[1]河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心,河北保定071001

出　　处：《中国农业科学》2013年第23期4926-4932,共7页Scientia Agricultura Sinica

基　　金：河北省高层次人才资助项目(20120346);国家"863"项目(2011AA10A205);教育部博士点专项基金(20121302120010)

摘　　要：【目的】马铃薯疮痂病菌具有明显的组成多样性,通过筛选不同疮痂病菌的通用探针引物,建立针对该类病原菌的定性检测方法。【方法】以目前中国存在的不同致病种的代表菌株CPS-1(Streptomyces scabies)、CPS-2(S.galilaeus)、CPS-3(S.acidiscabies)、CPS-4(S.turgidiscabies)为材料,根据NCBI GenBank中已登记的疮痂病菌特有毒素合成基因簇中的基因txtA、txtB、txtC(P450)、txtD(nos)等序列设计引物,对不同菌株的基因组扩增,选择特异性和稳定性较高的引物作为探针引物,经温度梯度筛选优化反应体系后建立成熟的检测技术。【结果】获得的通用检测引物B1/B2对所有致病菌株均表现较好的特异性,可稳定扩出目的条带,而非致病菌株均无条带。利用孢子稀释法验证建立的检测方法对菌株DNA的灵敏度为20 pg·μL-1,对孢子的检测阈值约为4.0 CFU/μL。对测试样品基因组扩增结果表明,疮痂病原链霉菌、病薯样品组织、病田土样均检测到目的条带,而非疮痂病原链霉菌、健康薯块组织、非病田土样和其它菌株均未扩出目的条带,说明该方法的特异性较好。【结论】建立了马铃薯疮痂病菌的定性检测方法,可实现对菌株、病组织和土壤样品的快速检测。[Objective] The composition diversity of common scab pathogens was confirmed by previous researchers. A qualitative detection method for potato scab pathogens was established in this work by screening the universal probe primers among different pathogens. [ Method ] Four typical pathogenic strains which belonged to Streptomyces scabies （CPS- 1）, S. galilaeus （CPS-2）, S. acidiscabies （CPS-3） and S. turgidiscabies （CPS-4） were selected to extract genome DNA. The primers were designed according to the sequences of four genes txtA, txtB, txtC （P450） and txtD （nos） that were from pathogen-specific toxin biosynthesis gene cluster what had registered in the NCBI GenBank. All primers were used for amplification for screening a high specificity and stability primers. After temperature gradient filter for optimizing the reaction system, a detection technology of potato scab pathogens was established. [Result] The universal primers B1/B2 obtained in this work showed a good specificity to all tested pathogenic strains. The stable bands were amplified among the pathogenic strains, but nonpathogenic strains didn＇t have that band. By using the spore dilution method, the sensitivity of PCR detection method was determined to 20 pg-~L1. The detection value of the threshold was 4.0 CFU/pL for pathogen spores. The PCR detection results showed that the target bars could be observed in pathogenic Streptomyces, scabby tissue and diseased field soil samples. While the nonpathogenic Streptomyces, healthy potato tissues, scab-free field soil and other test strains did not have corresponding objective band. That suggested the PCR detection method established in this work was specific and stable to common scab pathogens. [Conclusion] A qualitative detection method for potato scab pathogens was established in this study, which can quickly detect pathogenic strains from diseased tissues and soil samples.

关 键 词：马铃薯疮痂病菌 PCR检测 特异性引物 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]