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Published on: February 1, 2014
Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay
Sil Eun Kang1, Yong Suk Nam, Kwang Won Hong
1Department of Food Science and Technology, Dongguk University, Seoul 100-715, Korea.
Insights
A new real-time PCR method accurately detects Enterobacter sakazakii, an infant pathogen. This rapid, sensitive test identifies E. sakazakii in food, crucial for preventing infant meningitis and sepsis.
Area of Science:
- Microbiology
- Food Safety
- Molecular Biology
Background:
- Enterobacter sakazakii is a significant foodborne pathogen causing severe illness in infants.
- Infections like meningitis and sepsis in neonates have a high mortality rate.
- Contaminated infant formula is a primary transmission route.
Purpose of the Study:
- To develop a rapid, sensitive, and quantitative real-time PCR protocol for E. sakazakii detection.
- To utilize TaqMan probe technology based on 16S rRNA gene sequences.
- To ensure accurate identification of E. sakazakii in food safety applications.
Main Methods:
- Designed four primer sets and one TaqMan probe targeting the 16S rRNA gene.
- Tested the protocol against five E. sakazakii strains and 28 non-E. sakazakii strains.
- Determined the detection limits for bacterial cells and genomic DNA.
Main Results:
- The real-time PCR protocol successfully identified all tested E. sakazakii strains.
- No non-E. sakazakii strains were detected, confirming specificity.
- Achieved detection limits of 2.3 CFU/assay for cells and 100 fg/assay for DNA.
Conclusions:
- The developed TaqMan real-time PCR method is a reliable tool for E. sakazakii detection.
- This method offers high sensitivity and specificity for food safety monitoring.
- It provides a rapid and quantitative approach to control E. sakazakii contamination.
Abstract:
Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 16S rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.
