Dataset Information

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.

ABSTRACT: BACKGROUND: Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foods. Performance of the assay, coupled to an automated DNA extraction system and the E. sakazakii ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment. RESULTS: The real-time PCR assay had 100% specificity as assessed using 35 E. sakazakii and 184 non-E. sakazakii strains. According to the E. sakazakii strains tested, the detection limits ranged from 5 to 25 genomic copies. Assays on pure cultures (including real-time PCR and DNA extraction) gave a sensitivity of about 102 to 103 CFU/ml. Out of 41 naturally contaminated infant formulae and environmental samples analysed for the presence of E. sakazakii, 23 were positive by real-time PCR and 22 by the conventional culture method, giving 97.5% concordance with the ISO-IDF reference method. CONCLUSION: This method, combining specific real-time PCR, automated DNA extraction and ISO-IDF standard enrichments, provides a useful tool for rapid screening of E. sakazakii in food and environmental matrices.

SUBMITTER: Derzelle S

PROVIDER: S-EPMC1713243 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.

Derzelle Sylviane S Dilasser Françoise F

BMC microbiology 20061213

<h4>Background</h4>Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foo ...[more]

PMID: 17166252

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Project description:BackgroundWhile improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.FindingsThe performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.ConclusionThis study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.

Dataset Information

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.

Publications

A robotic DNA purification protocol and real-time PCR for the detection of Enterobacter sakazakii in powdered infant formulae.

Similar Datasets

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