| Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification method, which is simplicity, rapidity, high sensitivity and high specificity, and widely used in the research of nucleic acid,clinical diagnosis and the detection of genetically modified organism etc. It has the potential to replace PCR.Pantoea stewartii subsp. stewartii and Clavibacter michiganense subsp. Sepedonicum are two kinds of important quarantine bacterium, The traditional methods for the detection of Pantoea stewartii subsp. stewartii and Clavibacter michiganense subsp. sepedonicum were isolating culture, Physiological and biochemical identification, serological test and ELISA, which are time-consuming and low efficiency, and not apply for the port need. So it important to establish a rapid detection method of Pantoea stewartii subsp. stewartii and Clavibacter michiganense subsp. Sepedonicum.The specific region of the genome of Pantoea stewartii subsp. stewartii and Clavibacter michiganense subsp. sepedonicum were used for the design of LAMP primers, the LAMP detection system were established by optimization of reaction conditions. Specificity of the systems was verified using a series of reference strains, sensitivity of the systems were verified using a series of gradient dilution of DNA solutions of the Pantoea stewartii subsp. stewartii and Clavibacter michiganense subsp. sepedonicum,and the bacterial suspensions. In the specificity tests, LAMP systems showed high and identical specificity. DNA and bacterial suspension sensitivity of the Pantoea stewartii subsp. stewartii LAMP system reached0.858×10-4ng/uL and45CFU/mL respectively. DNA and bacterial suspension sensitivity of the Clavibacter michiganense subsp. sepedonicum LAMP system reached0.527×10-3ng/uL and150CFU/mL respectively. The two LAMP systems provide new, fast and easy way for the detection of Pantoea stewartii subsp. Stewartii and Clavibacter michiganense subsp. sepedonicum. |
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