Pcr-Based Techniques For Detection Of Xanthomonas Oryzae Pv. Oryzae And Xanthomonas Oryzae Pv. Oryzicola

Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola are important pathogens which cause rice disease. Bacterial blight and bacterial leaf streak of rice are quarantine pathogens in some countries in the world. It is very important to detect the bacteria pathogens rapidly and specifically for plant quarantine.Universal PCR primers Sr1 (5' -AGAGTTTGATCATGGCTCAG-3' ) and Sr2 ( 5 ' -ACGGTTACCTTGTTACGACTT-3 ' ) , L1 ( 5 ' AGTCGTAACAAGGTAGCCGT-3 ' ) and L2 ( 5 ' -GTGCCAAGGCATCCACC-3 ' ) . C1 ( 5 ' -ATTTCCGAATGGGGIAACCC-3 ' ) and C3 ( 5 ' -TGTCTCACGACGTTITAAACCCAGCTC-3' ) that target the 16S rDNA, ITS, 23S rDNA region were utilized in the assay. A similar fragment about 1.6kb, 600bp or 2.3kb was amplified from each of 3 tested strains by PCR. The PCR products were cloned and sequenced. We compared these sequence alignments with the homology array from the database on the internet and combined the published gene sequences of two bacteria pathogens , and then two pairs of specific primers were designed and synthesized. Primers were used to amplify the specific region from 67 strains of Xanthomonas oryzae pv. oryzae , 31 strains of Xanthomonas oryzae pv. oryzicola and 20 other tested bacteria strains and proved the specificity of this two pairs of primers was high. We developped the immuno-capture PCR method, by which could enhanced 10 times of sensitivity of detection pure culture.We setted up succeedly multiplex-PCR and optimize its reactive condition, by which we could detect Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola rapidly and specifically. A specific fragment about 1500bp was amplified from strains of Xanthomonas oryzae pv. oryzae by this multiplex -PCR; a specific fragment with 338bp was amplified from strains of Xanthomonas oryzae pv. oryzicola; two specific fragments with 1500bp and 338bp were amplified from the mixture of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola; but not other tested bacteria strains. It issimple and convenient and rapid because this multiplex -PCR amplify directly from pure culture; and its cost is low. So this multiplex -PCR technique is adapt to routine plant quarantine in port.