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

• Published in: International journal of food microbiology Vol. 118; no. 2; pp. 132 - 138
• Main Authors: Yang, Hua, Qu, Liangwei, Wimbrow, Adrienne N., Jiang, Xiuping, Sun, Yaping
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-09-2007; Elsevier
• Subjects: Animals; bacterial contamination; Biological and medical sciences; culture media; DNA, Bacterial - analysis; food analysis; food contamination; Food Contamination - analysis; Food industries; Food Microbiology; food pathogens; food safety; Fundamental and applied biological sciences. Psychology; General aspects; Humans; Immunomagnetic separation; Immunomagnetic Separation - methods; Listeria monocytogenes; Listeria monocytogenes - isolation & purification; Magnetic nanoparticles; Methods of analysis, processing and quality control, regulation, standards; microbial detection; milk; Milk - microbiology; Nanoparticles; nanotechnology; Polymerase Chain Reaction - methods; rapid methods; Real-time PCR; reverse transcriptase polymerase chain reaction; safety testing; Sensitivity and Specificity; Time Factors; Listeria monocytogenes; Immunomagnetic separation; Real-time PCR; Magnetic nanoparticles; Polymerase chain reaction; Analysis method; Separation; Bacteria; Control method; Molecular biology; Detection; Real time
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2007.06.019

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.