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


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.


CONCLUSIONS
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.
Amagliani
G
G
Centro di Biotecnologie, Università degli Studi di Urbino, Fano, Italy.
Omiccioli
E
E
Campo
A
A
Bruce
I J
IJ
Brandi
G
G
Magnani
M
M
D000327:Adsorption; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D015169:Colony Count, Microbial / Q000379:methods; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D005516:Food Microbiology; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D049329:Nanostructures; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*
D001427:Bacterial Toxins; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes