Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables
Language English Country England, Great Britain Media print-electronic
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
PubMed
25431169
DOI
10.1111/lam.12367
Knihovny.cz E-resources
- Keywords
- DNA isolation, Listeria monocytogenes, inhibition, real-time PCR, vegetable,
- MeSH
- DNA, Bacterial genetics isolation & purification MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Listeria monocytogenes genetics isolation & purification MeSH
- Listeriosis prevention & control MeSH
- Foodborne Diseases prevention & control MeSH
- Food Microbiology methods MeSH
- Vegetables microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Bacterial MeSH
UNLABELLED: Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes.
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