玉米内州萎蔫病菌的巢式PCR检测方法

Nested-PCR detection of Clavibacter michiganensis subsp. nebraskensis

目的建立了一种特异、敏感、快速的玉米内州萎蔫病菌的巢式PCR(Nest-PCR)检测方法。方法根据玉米内州萎蔫病菌(Clavibacter michiganensis subsp.Nebraskensis,Cmn)和C.michganensis种下其他4个亚种细菌Cmm、Cmi、Cms、Cmt的ITS序列差异性基因位点设计Cmn FP-OUTER/Cmn RP-OUTER为外侧引物,Cmn FP-INNER/Cmn RP-INNER为内侧引物的巢式-PCR,并对所有参试菌株DNA和菌悬液进行了扩增。结果经过两轮扩增,能从参试的玉米内州萎蔫病菌中特异性地扩增出1条122 bp的片段,而其他参试菌株没有扩增信号。该巢式PCR检测方法对菌悬液的最低检测限为3.54×102 CFU/m L,检测灵敏度比常规PCR提高了1000倍。结论本研究建立的玉米内州萎蔫病菌的巢式PCR(nest-PCR)检测方法具有良好的特异性和灵敏性,将会在玉米内州萎蔫病的早期诊断及预防传入方面发挥重要作用。

Objective This study is to develop a nested-PCR detection method, which is specific, sensitive and effective, to detectClavibacter michiganensis subsp.nebraskensis.MethodsAccording to the difference of gene loci of intergenic transcription spacer (ITS) areas ofClavibacter michiganensis subsp. nebraskensis(Cmn) and related species including Cmm, Cmi, Cms and Cmt, the nested-PCR with primers including CmnFP-OUTER/CmnRP-OUTER and CmnFP-INNER/CmnRP-INNER was designed, and the tested bacteria strain DNA and suspension were amplified.Results After two rounds of amplification, a band of 122 bp was amplified from Cmn strains but not from other tested bacteria species. The nested PCR detection method can detect the Cmn suspension with minimum limit of 3.54×102 CFU/mL, and the sensitivity of this method is 1000 times higher than conventional PCR method.ConclusionThe nested-PCR detection method developed in this study has a good specificity and sensitivity for detectingClavibacter michiganensis subsp. nebraskensis,and it will play an important role in early diagnosis and prevention of Goss′s bacterial wilt and blight of maize.