Establishment of RT-LAMP-HNB Method for Listeria monocytogenes Detection
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Abstract:
A rapid detection method RT-LAMP-HNB for Listeria monocytogenes was developed. Two pairs of primers were designed according to the conserved region of the hlyA gene sequence. In the same reaction system, the RNA of Listeria monocytogenes was reverse transcripted to cDNA and then was amplified by loop mediated isothermal amplification. At the same time, hydroxyl naphthol blue (HNB, the final concentration of 200 μM) was used as an indicator for reverse transcription loop mediated isothermal amplification products (RT-LAMP). The amplified results can be interpreted according to the color change of the system, and the gel electrophoresis was not necessary. This detection can be done within 20 hours (including the time of enrichment) and the live Listeria monocytogenes in milk can be detected. The detection limit in this study for the RNA of Listeria monocytogenes is 5.8×10-3 μg /mL, the detection limit of the initial inoculation concentration is 10 CFU/10mL, which are 10 times more sensitive than the RT-PCR method. RT-LAMP-HNB detection method developed in this study has the characteristics of fast, accurate, convenient and high sensitivity, which is suitable for popularization and application in grass-root labs or inconvenient areas.
