Abstract
A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting I CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.
| Original language | English |
|---|---|
| Journal | Applied and Environmental Microbiology |
| Volume | 70 |
| Issue number | 1 |
| Pages (from-to) | 69-75 |
| Number of pages | 7 |
| ISSN | 0099-2240 |
| DOIs | |
| Publication status | Published - 2004 |
| Externally published | Yes |
Keywords
- SALMONELLA
- Animal Feed
- Animals
- Buffers
- Colony Count, Microbial
- Culture Media
- DNA-Directed DNA Polymerase
- Polymerase Chain Reaction
- Salmonella
- Sensitivity and Specificity
- Species Specificity
- Time Factors
- EC 2.7.7.- Tth polymerase
- EC 2.7.7.7 DNA-Directed DNA Polymerase
- Amplification
- Bioassay
- DNA
- Probability
- Regression analysis
- Microbiology
- DNA polymerase
- animal food
- article
- cell viability
- colony forming unit
- controlled study
- cytolysis
- diagnostic accuracy
- DNA extraction
- early diagnosis
- evaluation
- food chain
- gene amplification
- intermethod comparison
- logistic regression analysis
- nonhuman
- polymerase chain reaction
- probability
- safety
- sensitivity analysis
- strain difference
- Animalia
- Negibacteria
- Culture enrichment
- X
- BIOTECHNOLOGY
- MICROBIOLOGY
- POLYMERASE-CHAIN-REACTION
- YERSINIA-ENTEROCOLITICA
- CONTAMINATED FEED
- AMPLIFICATION
- ASSAY
- IDENTIFICATION
- OPTIMIZATION
- PREVALENCE
- INFECTION
- ENTERICA
- animal feed samples animal feed, bacterial contamination, bacterial source, microbial analysis, safety
- food microbiology
- food safety
- Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Salmonella genus pathogen detection methods
- DNA polymerase 9012-90-2 EC 2.7.7.7
- 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
- 10802, Enzymes - General and comparative studies: coenzymes
- 13502, Food technology - General and methods
- 26504, Animal production - Feeds and feeding
- 31000, Physiology and biochemistry of bacteria
- culture enrichment techniques culturing techniques, laboratory techniques
- PCR polymerase chain reaction genetic techniques, laboratory techniques
- Foods
- Methods and Techniques
- ENRICHMENT
- FEEDS
- FOOD ENRICHMENT
- FOOD SAFETY ANIMAL FOODS
- GENETIC TECHNIQUES
- PCR
- General aspects
- Meat, poultry and game