摘要
[目的]建立玉米内州萎蔫病菌的快速恒温扩增检测技术。[方法]利用Gen Bank公布的玉米内州萎蔫病菌(Clavibacter michiganensis subsp.nebraskensis,Cmn)16S-23S序列设计LAMP(loop-mediated isothermal amplification)引物,建立玉米内州萎蔫病菌LAMP检测体系,并对反应条件进行了优化。[结果]LAMP在内引物、环化引物和外引物比为6∶4∶1(30 pmol∶20 pmol∶5 pmol)的25μl体系下64℃反应30 min为最佳反应条件,且LAMP引物能从供试的7种菌株中特异性扩增出Cmn梯度性条带。以玉米内州萎蔫病菌基因组和菌悬液梯度稀释液为模板检测LAMP体系的灵敏度,结果显示,LAMP检测体系针对基因组和菌悬液的灵敏度分别达到了50 fg和3.6CFU,比普通PCR灵敏度高100倍。[结果]LAMP检测体系简单、快速、灵敏度高、特异性好,在玉米内州萎蔫病菌检测方面潜力巨大。
[Objective]The aim was to establish a rapid detection method of Clavibacter michiganensis subsp. nebraskensis LAMP. [Method]The LAMP primers was designed by the rRNA 16S-23 S of the genome of Clavibacter michiganensis subsp. nebraskensis,were verified using Clavibacter michiganensis subsp. nebraskensis and other non-target strains. [Result]The data showed that the optimal amplification condition was at 64 ℃ for 30 min with 30 pmol of F3（ B3）,20 pmol of LF（ LB） and 5 pmol of FIP（ BIP）. Both the sensitivity and the specificity of the LAMP assay developed in the study were excellent. The detection limit of the LAMP was as low as 50 fg（ for DNA） and 3. 6 CFU（ for bacteria） per reaction. Compared with conventional PCR method,the LAMP assay developed in this study was more specific and more sensitive.Therefore,the LAMP method could be considered as an effective tool for the rapid screening of Clavibacter michiganensis subsp. nebraskensis.[Conclusion]The study showed that the loop-mediated isothermal amplification（ LAMP） could be used as a reliable,rapid and simple method for detection of Clavibacter michiganensis subsp. nebraskensis（ Cmn） causing Goss＇ s Bacterial Wilt and Leaf Blight of Corn.
出处
《安徽农业科学》
CAS
2015年第22期17-20,22,共5页
Journal of Anhui Agricultural Sciences
基金
浙江检验检疫局科研项目(ZK201227)
浙江省科技厅重大科技专项重点农业项目(2009C12054)
浙江省科技厅公益技术研究农业项目(2013C32002)
关键词
玉米内州萎蔫病菌
LAMP
检测
Clavibacter michiganensis subsp.nebraskensis
LAMP
Detection
