Isolation And Characterization Of Pseudomonas Aeruginosa From The Minks And Development Of Loop-mediated Isothermal Amplification Method

Hemorrhagic pneumonia of mink is an acute infectious disease caused byPseudo mo na s a e rugin o sa, clinical signs are various for bleeding from the nose ormouth, dyspnoea and sudden death.This infectious disease makes the mosteconomic loss for the farmed mink in China, therefore it is necessary to carry outthis study of the disease.P se ud o mo n a s a erugino sa is a common bacterium that can cause disease inanimals and humans. It is a Gram-negative, rod-shaped bacterium with unipolarmotility by monotrichate flagellum. Pseudomonas aeruginosa has strong resistanceto the external environment, and can survive in a polluted environment and soil fora long time, and is resistant to many chemical disinfectants and antibiotics.A Pseudomonas aeruginosa was isolated from the lung of a dead mink fromWeihai of Shan Dong Province.We identified the Pseudomonas aeruginosa basedon colony characteristics,biochemical tests and PCR. Pathogenicity test showedthat the LD50for mice is1.4×106.0CFU,and the MLD is8.0×105.0CFU.Wesuccessfully isolated the same bacteria as infectious bacteria from the infectiousmice.Drug susceptibility test showed that the isolated was sensitive to some drugssuch as gentamicin,Ciproflaxin and norfloxacin,but resistant to penicillin andstreptomycin. On the basis of the bacterial16srRNA gene sequence phylogeneticanalysis and comparison of this gene sequence with sequence in RNA sequencedatabase, it was considered that the isolated was closely related to me mbers of thePseudo mo na s aerugino sa from the wheat,so we suepected that the isolated wasfrom the contaminated feed.LAMP is a rapid and visible method to detect the Pseudomonas aeruginosa.Aset of four primers were designed based on the PA-16SrDNA gene sequence byPrimerExplorerV4software. The specificity and sensitivity are very good byoptimizing reaction system and reaction time.It was showed that the Pseudomonsaaeru ginoa s could be detected within60minutes by this LAMP, and the sensitivitywas100times higher than that of general PCR. Establishment of LAMP provides anew alternative method for the rapid detection of Pseudomonsa aeruginoas,and hasa good prospect in the labortary and clinical tests.