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Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples

Amagliani, G., Omiccioli, E., Del Campo, A., Bruce, Ian J., Brandi, G., Magnani, M. (2006) Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples. Journal of Applied Microbiology, 100 (2). pp. 375-383. ISSN 1364-5072. (doi:10.1111/j.1365-2672.2005.02761.x) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:71954)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1111/j.1365-2672.2005.02761.x

Abstract

Aims: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. Methods and Results: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml-1 contamination rate. Conclusions: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. Significance and Impact of the Study: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.

Item Type: Article
DOI/Identification number: 10.1111/j.1365-2672.2005.02761.x
Uncontrolled keywords: bacterial DNA; nucleic acid, polymerase chain reaction, analytic method; article; bacterial gene; bacterium contamination; bacterium detection; comparative study; DNA purification; food contamination; hlya gene; hybridization; Listeria monocytogenes; magnetic capture hybridization; magnetic separation; milk; nanoparticle; nonhuman; polymerase chain reaction; process development; reproducibility; sensitivity and specificity; technique, Adsorption; Animals; Bacterial Toxins; Colony Count, Microbial; Culture Media; DNA Probes; DNA, Bacterial; Food Microbiology; Heat-Shock Proteins; Hemolysin Proteins; Listeria monocytogenes; Milk; Nanostructures; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Polymerase Chain Reaction, Bacteria (microorganisms); Listeria monocytogenes
Subjects: Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Physical Sciences
Faculties > Sciences > School of Physical Sciences > Functional Materials Group
Depositing User: Ian Bruce
Date Deposited: 01 Feb 2019 12:49 UTC
Last Modified: 30 May 2019 08:49 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/71954 (The current URI for this page, for reference purposes)
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