A Real-time Fluorescence Loop-mediated Isothermal Amplification Method to Detect Enterobacter sakazakii in Milk Powder
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Abstract:
In order to rapidly detect Enterobacter sakazakii in milk powder, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) method was developed. Three sets of loop-mediated isothermal amplification (LAMP) primers targeting the Enterobacter sakazakii 16S rRNA sequence were designed. With a DEAOU-308C thermostat fluorescence detector, the specificity of the primers was examined using a standard strain of Enterobacter sakazakii. The sensitivity and detection limit on genomic DNA were also determined with a standard strain of Enterobacter sakazakii, followed by measuring the sensitivity and detection limit of this assay for artificially contaminated skimmed milk powder and whole milk powder, and a comparative experiment was performed on 20 commercial milk powder samples using the RealAmp method and national standard method. The results showed that the primer set 16S-11 had the best amplification efficiency, showed no cross-reactivity with common pathogens, and the sensitivity of RealAmp for detecting Enterobacter sakazakii genomic DNA, artificially contaminated skimmed milk powder, and whole milk powder reached 102 CFU/mL, 102 CFU/mL, and 103 CFU/mL, respectively. The detection limit of RealAmp in detecting Enterobacter sakazakii genomic DNA, artificially contaminated skimmed milk powder, and whole milk powder reached 103 CFU/mL, 103 CFU/mL ,and 104 CFU/mL, respectively. The results of 20 commercial milk powder samples obtained by the RealAmp method were consistent with those from the national standard culture method. The results suggest that this RealAmp assay is suitable for the rapid detection of Enterobacter sakazakii in milk powder.
