番茄溃疡病菌分子检测技术

Development of a PCR Assay for the Detection of Clavibacter michiganensis subsp. michiganensis

对中国三类检疫性有害生物番茄溃疡病菌(Clavibactermichiganensissubsp.michiganensis)进行PCR检测方法的研究。利用1对特异性引物ClaF1ClaR2,对5个番茄溃疡病菌进行特异性扩增,得到了一段长250bp的PCR产物,参试的其他棒形细菌以及其他属的植物病原细菌均无扩增产物。该检测方法特异性强、灵敏度高,在50pg的模板DNA浓度下还能检测到很强的条带。利用此引物可以检测到1×105个菌体。

Clavibacter michiganensis subsp. michiganensis is a seed-borne bacterium listed as an A 3 pest in China. A pair of primers ClaF1-ClaR2 was deduced and a PCR protocol was developed for Clavibacter michiganensis subsp. michiganensis-specific amplification of a 250 bp ITS(intergenic spacer region)fragment when Cmm genomic DNA was used as template. No DNA was amplified from phenotypically related bacteria, including species of Curtobacterium, Arthrobacter, Rathayibacter, Rhodococcus and pathovars of Clavibacter and other pathogenic bacteria by the same primers. The PCR assay was proved to be sensitive for detection of Cmm sufficiently in 50 pg template DNA or 1×10~5 cfu strains. It was demonstrated to be a highly sensitive, specific and rapid PCR assay for the detection of Cmm.