Document Detail

Real-time PCR detection of 16S rRNA genes speeds most-probable-number enumeration of foodborne Listeria monocytogenes.
MedLine Citation:
PMID:  17685338     Owner:  NLM     Status:  MEDLINE    
Quantifying foodborne pathogens at concentrations of 0.1 to 1,000 CFU/g of food generally involves most-probable-number (MPN) enumeration, which takes at least 4 days. A real-time PCR assay (RTi-PCR) was developed to accelerate MPN enumeration of foodborne Listeria monocytogenes. Foods were spiked from 70 to 110 CFU/g, and triplicate subportions from 0.0001 to 1 g were selectively enriched for 48 h at 30 degrees C. For standard MPN enumeration, the enrichments were subcultured on Oxford agar (48 h at 35 degrees C) to isolate Listeria. For RTi-PCR MPN, the L. monocytogenes cells from the same enrichments were washed and resuspended in 2 ml of sterile water. DNA was extracted by boiling for 10 min. The DNA in the extract's supernatant was targeted with published oligonucleotide primers for amplifying an Lmo-specific sequence of 16S rRNA genes. Amplification was continuously monitored with SYBR Green. The resulting amplicon was characterized by its melting temperature. The L. monocytogenes specificity of the primers was confirmed by testing L. monocytogenes (15 strains), Listeria innocua (11 strains), and Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, and Listeria grayi (1 strain each). Quantitatively spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork pâté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the standard and the PCR MPN method. The paired results from the two MPN methods agreed well, except for the fresh cheese. For some foods, l-g samples required a decimal dilution for a positive test result, suggesting concentration-dependent food ingredient interference with the RTi-PCR. This RTi-PCR method reduced the time necessary for the MPN enumeration of foodborne L. monocytogenes from 4 to 2 days.
Elaine Cristina Pereira De Martinis; Robert E Duvall; Anthony D Hitchins
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of food protection     Volume:  70     ISSN:  0362-028X     ISO Abbreviation:  J. Food Prot.     Publication Date:  2007 Jul 
Date Detail:
Created Date:  2007-08-09     Completed Date:  2007-09-17     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7703944     Medline TA:  J Food Prot     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1650-5     Citation Subset:  IM    
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil.
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MeSH Terms
Bacteriological Techniques / methods
Cheese / microbiology
Colony Count, Microbial / methods*
Dairy Products / microbiology
Food Contamination / analysis*
Food Microbiology*
Listeria monocytogenes / isolation & purification*
Meat Products / microbiology
RNA, Bacterial / genetics
RNA, Ribosomal, 16S / genetics
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Reg. No./Substance:
0/RNA, Bacterial; 0/RNA, Ribosomal, 16S

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