Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR

Abstract

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti- L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles ( C T) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads ®-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes ≥ 10 2 CFU/0.5 ml. In the range of 10 3 to 10 7 L. monocytogenes CFU/0.5 ml, cell numbers calculated from C T values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti- L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.