Development of cell surface protein associated gene probe specific for Listeria monocytogenes and detection of the bacteria in food by PCR.
A genomic library of L. monocytogenes was constructed using lambda Zap II-Eco RI and screened with a monoclonal antibody which is specific for a Listeria cell surface protein. Three positive clones each contained a 6.5 kb insert which in E. coli could express the same Listeria protein. The 6.5 kb insert was further digested with Hin dIII and the smaller fragments were subcloned into a plasmid vector (pBluescript) and screened with 32P-labelled genomic DNA from L. monocytogenes or L. innocua. Three clones which were positive with L. monocytogenes and negative with L. innocua were screened and each contained a 2.1 kb insert. The 2.1 kb insert was partly sequenced and some candidate oligomer probes from the sequences were selected and compared with sequences in a Genbank computer search. One such oligomer probe (T7-list) was confirmed to be specific for L. monocytogenes. The probe hybridized with all 28 strains of L. monocytogenes tested, but not with any of six other Listeria species nor 11 other bacteria tested. Using this probe-primer, a PCR method was developed which could detect as few as 2 cfu of L. monocytogenes in pure cultures, and as few as 4-10 cfu of L. monocytogenes when inoculated into foods.
Wang
R F
RF
Department of Food Science and Arkansas Biotechnology Center, University of Arkansas, Fayetteville 72703.
Cao
W W
WW
Johnson
M G
MG
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000302:isolation & purification; D003845:Deoxycytosine Nucleotides; D005516:Food Microbiology; D015698:Genomic Library; D015151:Immunoblotting; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D003845:Deoxycytosine Nucleotides; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; C024107:2'-deoxycytidine 5'-triphosphate