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.

Real-Time PCR Method Using Capturing Oligo-Immobilized PCR Tubes to Determine the Specific Gene for Soybean and Genetically Modified Soybean in Food Matrices

“…In previous reports, the hybridization times were 2 h for a one-step system and 4 h for a two-step system. 1,10) Our data show that this method can be carried out with a relatively short hybridization time. We subsequently used 30-min hybridization in this study.…”

“…1,[10][11][12] We tested an incubation temperature from 25 to 60 C. The copy number of Le1 in an Le1n02-3 0 -a-immobilized tube was successfully detected at 40 to 65 C, especially at 50 C, but detection at 25 C was poor (25 C, 40 AE 5 copies; …”

“…The bead method can reduce the total detection time, remove inhibitors of the PCR amplification reaction, and remove excess nontarget DNA. 1,2) However, the hybridization bead method is not used directly in real-time PCR as the beads block the optical path. Ideally, the number of tube-to-tube transfers should be kept to a minimum to avoid the loss of template and decrease the risk of contamination.…”

Review. Specific detection of Listeria monocytogenes in foods using commercial methods: from chromogenic media to real-time PCR

“…The amplification performed with the purified DNA could reliably identify 10 cells ml -1 , a detection level more sensitive than the PCR carried out with nucleic acids obtained using commercial nanoparticles. The method avoids the pre-enrichment and provides a rapid alternative to conventional microbiological detection methods (Amagliani et al, 2006).…”

Detection of foodborne pathogens using bioconjugated nanomaterials

“…Amagliani et al (2006) immobilized oligonucleotide probes to magnetic nanoparticles for selective DNA purification. Followed by PCR, L. monocytogenes cells were detected from milk samples at a 10 CFU/ml contamination rate.…”