Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.
A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.
Fluit
A C
AC
Eijkman-Winkler Laboratory for Medical Microbiology, University Hospital Utrecht, The Netherlands.
Torensma
R
R
Visser
M J
MJ
Aarsman
C J
CJ
Poppelier
M J
MJ
Keller
B H
BH
Klapwijk
P
P
Verhoef
J
J
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins; D001483:Base Sequence; D002611:Cheese / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics; D005069:Evaluation Studies as Topic; D005516:Food Microbiology; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes