| The Phytobacteria, which can cause devastating disasters and great loss, are very harmful pathogens. Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis are two typical pathogens parasitized on corn, while neither of them has been detected in china. They are mainly spreaded by seed-borne and insect-borne. Owning to a great deal of imports of corn seeds from aboard, developing effective and efficient methods for quick detection has become gradually significant.Because of the high technical requirements of the detection for Phytobacteria, methods for the detection have been always the constraints in Port Plant Quarantine. Therefore, in order to effectively control the invasion of bacterial disease and improve detection technology of port pathogen, it is much important to develop a fast, simple and highly sensitive detection method.In this paper, bear beads and beads with different modifiers on the surface have been synthesized by coprecipitation technique and characterized with TEM and DLS test. PCR 16S rDNA amplification and sequencing have been adopted to preliminarily detect Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis. After incubation, the cause of the combination of beads and pathogens has been analysized. According to the non-specific binding of the beads with pathogens in culture solution, best beads are chosen, and reaction conditions of incubation, such as incubation temperature, time and the amount of beads, are optimized. Beads are incubated with pathogen solution under optimal condition and separated by magnetic power, and specific primers are used to specifically amplificate 16S rDNA products of target pathogens, enriched by magnetic beads, with PCR technique. SEM photographs of the two pathogens have been shot for the study of their morphology. The effect of beads on the toxicity of target pathogens has been detected by bacteriostasis test.Zeta potentials of the synthesized beads detected by DLS induced that different charges were on the surface of the beads and ones modified by PDDA carried positive charges, ones synthesized by co-precipitation negative charges, and ones modified by PSS and- COOH groups on the surface were with a strong negative charges. Particle size of beads synthesized ranges from 20 nm to 500 nm. PCR 16S rDNA specific amplification and sequencing alignment identified that the designed primers showed better specific. After incubation of four beads with pathogen solution, followed by analyzing the cause of combination of them, it was shown that beads with positive charge on the surface had the strongest power in the adsorption for the pathogens. Size and shape of beads had also an impact on the combination with pathogens. The absorption rate is proportional to the amount of used beads and beads balance at the concentration of 4 mg/mL. Probably because the best growth temperature for the target pathogens in this experiment is 24-28℃, temperature had little effect on absorption rate and so room temperature is advisable. Incubation time had much great effect on the absorption rate, with largest combination of Pantoea stewartii subsp. stewartii and beads at 40min after incubation and largest combination of Clavibacter michiganensis subsp. nebraskensis and beads at 20 min after incubation. The reason may be resulted from the differences in cell wall thickness and size of the two pathogens. After combination of pathogens with beads, they could be separated simply and quickly by magnetic power and pathogens in the solution could be condensed as well.After pathogens and beads incubation under the optimal condition, pathogens are condensed and the target pathogens can be specifically detected followed by PCR amplification. Thus it is concluded that the combination of magnetic beads absorption and PCR identification together can improve detection technique with simplicity, quickness and sensitivity for the application of pathogens quarantine. |
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