Nested PCR For Ultrasensitive Detection Of The Potato Ring Rot Bacterium

There is latent infection of Clavibacter michiganensis subsp. sepedonicus in potato tissues . The latent infection accumulates for generations and shows obvious symptoms finally. It is the basic reason that the ring rot of potato can't be eliminated thoroughly. So it is essential to detect the tiny quantity Clavibacter michiganensis subsp. sepedonicus in potato tissues. In order to find a rapid, accurate, sensitive and reliable method of molecular detection of Clavibacter michiganensis subsp. sepedonicus we refered to many materials and decided to study using Nested PCR to detect Clavibacter michiganensis subsp. sepedonicus.We first prepared the primer pairs CMSIF1- CMSIR1 and CMSIF2-CMSIR2, designed based on the sequence of the 1.3kb insertion element IS 1121 , a high repeated segment of DNA that is present in plasmid pCSl and in the chromosome of Clavibacter michiganensis subsp. sepedonicus, reported successfully. Formamide was added to the reaction mixture because the content of G and C of the primer pairs CMSIF1- CMSIR1 and CMSIF2-CMSIR2 is bigger . The results show that formamide can reduce the mismatching rate and increase the specific fragment production. The genomic DNA and suspension of Clavibacter michiganensis subsp. sepedonicus were amplified with the primer pairs CMSIF1- CMSIR1 separately and both had specific products (direct PCR amplified the 1.0kb fragment). So we can simplify the extraction of the genomic DNA of Clavibacter michiganensis subsp. sepedonicus. The suspension of Clavibacter michiganensis subsp. sepedonicus and the product of the direct PCR were amplified with the primer pair CMSIF2-CMSIR2 , but onlythe latter had a 0.9kb fragment product.Direct PCR and nested PCR were performed with the standard strains of Clavibacter michiganensis subsp. sepedonicus, the strains of Clavibacter michiganensis subsp. sepedonicus in Heilongjiang and the other main pathogenic bacteria in potato and only the strains of Clavibacter michiganensis subsp. sepedonicus had specific products (direct PCR amplified the 1.0kb fragment and nested PCR amplified the 0.9kb fragment).To compare the sensitivities of direct PCR and nested PCR a serial dilution of the suspension of Clavibacter michiganensis subsp. sepedonicus was amplified with the primer pair CMSIF1-CMSIR1 and the direct PCR products were used as templates in nested PCR with the primer pair CMSIF2-CMSIR2. The sensitivity of nested PCR increased 100- to 1000-fold. At the same time we got the results of the sensitivity of ELISA.The symptomatic tubers, the symptomless tubers and the healthy tubers were sampled for direct, PCR and nested PCR. The results were that all the samples of the symptomless tubers were detected positive.