Detection of Pseudomonas in Pork by Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method
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Abstract:
In this work, a real-time fluorescence loop-mediated isothermal amplification (LAMP) assay was established to detect Pseudomonas in pork. Based on the published 16S rDNA gene sequence, the 16S rDNA genes of 62 strains of Pseudomonas were sequenced by DNAMAN software to obtain the consensus fragment, which was used to design primers. The saturated nucleic acid dye Eva Green was added to the reaction system, which allowed amplification products to be measured by real-time fluorescence platforms. The amplification system was optimized and the optimization results were identified by electrophoresis. The specificity and sensitivity was compared with ordinary LAMP, and the detection limit of artificially contaminated pork was determined. The results showed that 12 strains of Pseudomonas are positive in the specific verification and 23 strains of non-Pseudomonas are negative. The sensitivity for Pseudomonas is 36 CFU/mL in pure cultures, which is 10 times more sensitive than the ordinary LAMP. The detection limit of artificially contaminated samples is 1.73×103 CFU/g. In this work, a method for detecting Pseudomonas in refrigerated pork was established, which can simultaneously detect Pseudomonas spp. and avoid the limitations of single species detection. The real-time fluorescent LAMP detection technology established in this work is less time-consuming, accurate and has high specificity and sensitivity. Pseudomonas in fresh pork could be detected within 2 hours by this method.
