Detection of Listeria monocytogenes by real time PCR assay using light upon extension fluorogenic primer
2006
Hiwaki, H.(Fukuoka-shi. Inst. for Hygiene and Environment (Japan)) | Baba, A. | Ebuchi, S. | Miyamoto, T.
A real-time quantitative PCR assay using light upon extension fluorogenic primer (LUX-qPCR) was designed to detect the hly gene of Listeria monocytogenes. LUX-qPCR was performed at 58 deg C annealing temperature with the primer sets of 2773FL/2882RU and 916FL/970RU. The assay was 100% specific for L. monocytogenes with the detection limit of 3,000 CFU/ml (15 CFU/reaction mixture), and the coefficient (r2) for the correlation between the amount of DNA and Ct value was calculated to be more than 0.99. The Listeria enrichment broth of the food specimens from which the L. monocytogenes strain was isolated was examined for the hly gene by LUX-qPCR with the 916FL/970RU primer set. The results of the culture method and LUX-qPCR in qualitative tests (25g sample enrichment test) were corresponding; however, in quantitative tests (MPN 3-tube test), the bacterial count obtained from LUX-qPCR was slightly larger than from the culture method. By improving the protocol of DNA template preparation and isolation of L. monocytogenes, both types of results will correspond better. In conclusion, LUX-qPCR was proved to be a rapid and useful molecular assay for the detection of the hly gene in food.
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