Direct detection of Listeria monocytogenes from milk by magnetic based DNA isolation and PCR

Two nucleic acid-based methods for rapid and sensitive detection of the foodborne pathogen Listeria monocytogenes in milk were developed in this study. These methods rely on paramagnetic nanoparticle-based isolation of bacterial DNA directly from milk and subsequent PCR with selective primers for the listeriolysin O (hlyA) gene. The hlyA specific product was reproducibly detected and showed a sensitivity of 10 cfu ml(-1). The magnetic-based system had a sensitivity 10-fold higher than that of commercially available column devices. The detection limit of both methods is sufficient for direct detection of L. monocytogenes DNA in milk avoiding the enrichment culturing step, reducing the time necessary to obtain results from samples to 7 h rather than the 5-day minimum required for the standard procedure. The methods developed are suitable for automation.

[1]  L. Cocolin,et al.  A simple and fast PCR protocol to detect Listeria monocytogenes from meat , 1997 .

[2]  W. Choi,et al.  Rapid enumeration of Listeria monocytogenes in milk using competitive PCR. , 2003, International journal of food microbiology.

[3]  G. Stewart,et al.  Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR) , 1992, Letters in applied microbiology.

[4]  F. Tjerneld,et al.  Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method , 1994, Applied and environmental microbiology.

[5]  L. Jaykus,et al.  Detection of Listeria monocytogenes from a Model Food by Fluorescence Resonance Energy Transfer-Based PCR with an Asymmetric Fluorogenic Probe Set , 2003, Applied and Environmental Microbiology.

[6]  I. Bruce,et al.  Magnetizable Solid‐phase Supports for Purification of Nucleic Acids , 1996, The Journal of pharmacy and pharmacology.

[7]  M. Wagner,et al.  Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay. , 2001, Research in microbiology.

[8]  N. S. Bansal.  Development of a polymerase chain reaction assay for the detection of Listeria monocytogenes in foods , 1996, Letters in applied microbiology.

[9]  P. Levison,et al.  Recent developments of magnetic beads for use in nucleic acid purification. , 1998, Journal of chromatography. A.

[10]  R. Moermans,et al.  Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers , 1995, Applied and environmental microbiology.

[11]  U. Stahl,et al.  Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction , 1998 .

[12]  S. Makino,et al.  A new method for direct detection of Listeria monocytogenes from foods by PCR , 1995, Applied and environmental microbiology.

[13]  J. L. Smith,et al.  Occurrence of Listeria in foods: milk and dairy foods. , 1990 .

[14]  J. Verhoef,et al.  Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay , 1993, Applied and environmental microbiology.

[15]  A. Schuchat,et al.  Epidemiology of human listeriosis , 1991, Clinical Microbiology Reviews.

[16]  L. Mascola,et al.  The epidemiology of listeriosis in the United States--1986. Listeriosis Study Group. , 1991, American journal of epidemiology.

[17]  K. Jinneman,et al.  Listeria monocytogenes Lineage Group Classification by MAMA-PCR of the Listeriolysin Gene , 2001, Current Microbiology.

[18]  C. Hurst,et al.  Application of magnetite and silica-magnetite composites to the isolation of genomic DNA. , 2000, Journal of chromatography. A.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  A. Holck,et al.  Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk , 2000, Applied and Environmental Microbiology.

[21]  M. Griffiths.  Listeria monocytogenes: Its importance in the dairy industry , 1989 .

[22]  K. Holmstrøm,et al.  Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. , 1992, International journal of food microbiology.

[23]  R. Díaz,et al.  Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle , 1995, Journal of clinical microbiology.

[24]  F. Baquero,et al.  Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes , 1988, Infection and immunity.

[25]  M. Magnani,et al.  High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein. , 2003, Protein expression and purification.

[26]  J Bickley,et al.  Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions , 1996, Letters in applied microbiology.

[27]  Bruce Ij.  Nucleic acid amplification mediated microbial identification. , 1993 .