Establishment Of Loop-mediated Isothermal Amplification Method In Detection Of Aeromonas Hydrophila

A novel nucleic acid amplification method, termed loop-mediated isothermalamplification (LAMP), which amplifies DNA with high specificity, efficiency, andrapidity under isothermal conditions, may be a valuable tool for the rapid detection ofinfectious diseases. This method employs a DNA polymerase that have activity ofstrand displacement DNA synthesis and a set of four specially designed primers thatrecognize a total of six distinct sequences on the target DNA. LAMP can amplify afew copies of DNA to 10~9 in less than an hour. The final products are stem-loop DNAwith several inverted repeats of the target and cauliflower-like structures with multipleloops. A positive reaction would be shown as a ladder-like pattern in a gelelectrophoresis analysis. Because of the advantage, the LAMP method will be widelyapplied to research of nucleic acid, clinical diagnosis of infectious diseases anddetection of genetically modified organisms etc.Three pairs of primers, designed from known sequences of aerolysin gene, areused to amplify fragments of the same gene from aeromonas hy.drophila with thegenome DNA as template by loop-mediated isothermal amplification. The finalproducts are stem-loop DNA with several inverted repeats of the target andcauliflower-like structures with multiple loops.We have established the new method of detection of aeromonas hy.drophilasuccessfully. As a result, The best temperature of LAMP is sixty four degree. LAMPcan detect thirty-eight fg/tube, and PCR can detect thirty-eight pg/tube from DNAsample. LAMP can detect thirty-two cell/tube, but PCR can detect nothing from thebacterial sample. LAMP is one thousand times sensitive than PCR.We also designed a pair of loop primers which can improve the speed, and thewhole time of detection is shorten to half an hour. This method will satisfy with thedetection of aeromonas hy. drophila.