Detection of <i>Listeria monocytogenes</i> in Foods by SYBR Green I Melting Curve

Abstract

Two pairs of specific primers were composited based on the hlyA gene of L.monocytogenes and the 16sRNA gene of the genus Listeria. Optimal reaction system and condition of the SYBR GreenⅠfluorescent PCR were established, respectively, which could detect two specific genes. The reaction solution was verified by the method of gel electrophoresis. The results showed that two target fragments possessed different sizes and Tm valves which were amplfied corresponding two separate peaks. The standard curve based on the L.monocytogenes hlyA gene was established and the correlation coefficient was 0.996. The minimal detection limit was 89CFU/mL. Moreover, suspicious strain isolated from white clams was tested by using this method so as to study the feasibility. The positive result was evaluated with the National Standard Methods (GB 4789.30-2010). The verified result indicated that the positive result of SYBR GreenⅠfluorescent PCR matched with the GB 4789.30-2010 exactly.

Presence of Listeria monocytogenes in Ready-to-Eat Artisanal Chilean Foods

“…The genomic DNA of the suspected strains was extracted and purified with an UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, Qiagen, Carlsbad, CA, USA). Strains were confirmed as L. monocytogenes by hlyA gene amplification [19] with the Stratagene Mx3000P real-time PCR System equipment (Agilent Technologies, Santa Clara, CA, USA) using the commercial Brilliant II SYBR Green QRT-PCR Master Mix [20] (Agilent Technologies, Santa Clara, CA, USA). Amplified products were sent for sequencing to Macrogen, Korea.…”