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

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