Development Of Duplex PCR Assay And Competitive PCR Assay For Detecting Of Clavibacter Michiganensis Subsp. Sepedonicus And Pectobacterium Atroseptica

Potato is the fourth food crop in the world, which is behind rice, wheat and corn. China is the world's biggest producer of potato. Potato ring rot (Clavibacter michiganensis subsp. Sepedonicus, Cms) and Potato blackleg (Pectobacterium atroseptica ,Eca) are the two important seed transmitted bacterial disease which impact on the potato production. Fast, accurate, sensitive and reliable detection of these two diseases is of great significance on reducing the percentage of infected seed tubers and the occurrence of the diseases. A duplex PCR assay for detecting of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica and a competitive PCR assay for quantification of Pectobacterium atroseptica were established. The results were as follows.1. Choosing the cellulose A gene sequence encoded by the native plasmid pCS1 of C. michiganensis subsp. sepedonicus which was published on the GenBank and comparing it with the nucleotide sequence of closely-related species and some pathogens of potato, a specific pair of primers, CMS1/CMS2, was designed and synthesized. Using CMS1/CMS2 primers, a single unique PCR band of 913 bp was amplified from C. michiganensis subsp. sepedonicus. The detection sensitivity was 100fg/μL of DNA and 10~5 CFU/mL of bacteria.2. A duplex PCR technology had been established under the optimized PCR parameters using the combining primers CMS1/CMS2 and ECA1f/ECA2r which was a specific pair of PCR primers to detect Pectobacterium atroseptica. Under the duplex PCR system, the 913 bp PCR band from Clavibacter michiganensis subsp. sepedonicus and 690 bp PCR band from P. atroseptica could be specifically amplified. The detection sensitivity was 600fg/μL of DNA and 5×10~5CFU/mL of bacteria. The duplex PCR technology also can be used to detect the tuber suffered from Clavibacter michiganensis subsp. sepedonicus and(or) Pectobacterium atroseptica and achieved good results. The two pathogens could be simultaneously and rapidly detected by the duplex PCR system.3. Eca genomic DNA was amplified using Eca specific primers ECA1f/ECA2r. The amplification product was cloned and sequenced. Using the DNA sequence of the amplification product and the sequences of specific primers ECA1f/ECA2r, a new forward primer, ECA3f, was designed and synthesized. Eca genomic DNA was amplified using new specific primers ECA3f/ECA2r, the amplification product is competitor PCR template. The competitor template was cloned into pEASY-T1 Clong Vector and transformed into Trans1-T1 competent cell, and internal control (E.coli 3f) was constructed for competitive PCR. Predetermined numbers of E.coli 3f were added to sterile water or potato peel extract both of which have Eca, and Eca numbers estimated by comparing the ratio of products generated Eca and E.coli 3f following PCR.