Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA).
A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10(2)-10(7) cfu cm-2. The detection threshold for the PCR-ELISA assay developed in this work is 10(2) cfu cm-2.
Departamento de Nutrición y Bromatología III, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain. RMARTIN@eucmax.sim.ucm.es
R
R
Gutiérrez
T
T
García
I
I
González
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B
Sanz
P E
PE
Hernández
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R
Martín
D001419:Bacteria / Q000302:isolation & purification*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology*; D008462:Meat-Packing Industry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S