Evaluation of paramagnetic beads coated with recombinant Listeria phage endolysin-derived cell-wall-binding domain proteins for separation of Listeria monocytogenes from raw milk in combination with culture-based and real-time polymerase chain reaction-based quantification.
The aim of this study was to evaluate a fast and simple bead-based method using paramagnetic beads covered with recombinant Listeria phage endolysin-derived cell-wall-binding domain proteins specific for Listeria spp. for separation of the foodborne pathogen Listeria monocytogenes from artificially contaminated raw milk. The method was combined with subsequent detection and quantification by the traditional plate-count technique and real-time polymerase chain reaction (PCR). To account for differences in cell properties, recovery rates and the detection limit were determined using five different L. monocytogenes strains for preparation of a 10-fold dilution series in raw milk, spanning an 8-log scale. Two independent test series were performed for each strain, yielding mean recovery rates of 46.6% to 122.8% for detection with the plate-count method, and 64.7% to 95.1% for detection by real-time PCR. A high correlation was found between the number of L. monocytogenes added to the samples and the number of colony forming units recovered by plate count (0.980), as well as the number of bacterial cell equivalents obtained by real-time quantitative PCR (0.987). The detection limit of the combined cell-wall-binding domain proteins/real-time PCR approach ranged from 10(2) to 10(3) colony forming units per milliliter, which is close to the theoretical detection limit of the method.
Walcher
Georg
G
Milk Technology and Food Science, Department of Farm Animals and Veterinary Public Health, Institute for Milk Hygiene, University of Veterinary Medicine, Vienna, Austria.
Stessl
Beatrix
B
Wagner
Martin
M
Eichenseher
Fritz
F
Loessner
Martin J
MJ
Hein
Ingeborg
I
D000818:Animals; D058491:Bacterial Load; D001431:Bacteriological Techniques; D001435:Bacteriophages / Q000201:enzymology; D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008863:Microspheres; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D014764:Viral Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C001143:endolysin