Establishment Of Loop-mediated Isothermal Amplification Method In Detection Of Xanthomonas Oryzae Pv.Oryzae

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious diseases. This method employs a DNA polymerase that have activity of strand displacement DNA synthesis and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. LAMP can amplify a few copies of DNA to 10⁹ in less than an hour. The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops. A positive reaction would be shown as a ladder-like pattern in a gel electrophoresis analysis. Because of the advantage, the LAMP method will be widely applied to research of nucleic acid, clinical diagnosis of infectious diseases and detection of genetically modified organisms etc.A set of primers, designed from known sequences of AF169030 , are used to amplify fragments of the same gene from Xanthomonas oryzae pv.oryzae with the genome DNA as template by loop-mediated isothermal amplification. The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops.We have established the new method of detection of Xanthomonas oryzae pv.oryzae successfully. When the reaction mixture containing 40 pmol each of FIP/BIP,10 pmol each of F3/B3,4mmol/L Mg²⁺ ,and incubating at 63℃for 60min,it can detect sample with high sensitivity. LAMP can detect forty-six pg/tube from DNA sample, by contrast, PCR can only detect four point six ng/tube from DNA sample. LAMP is one hundred times sensitive than PCR. Besides, LAMP can detect DNA from the bacterial sample, but PCR can detect nothing from the bacterial sample.