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PCR and oligonucleotide array for detection of Enterobacter sakazakii in infant formula
1Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Weijin Road 94, Tianjin 300071, China.
Insights
New PCR and oligonucleotide array methods rapidly detect Enterobacter sakazakii, a cause of neonatal meningitis. These reliable assays identify the bacteria in 48 hours, significantly faster than traditional methods.
Area of Science:
- Microbiology
- Molecular Biology
- Food Safety
Background:
- Enterobacter sakazakii is a pathogen linked to neonatal meningitis with high mortality rates.
- Accurate and rapid detection of E. sakazakii is crucial for public health, especially in infant formula.
- Conventional detection methods are time-consuming, often requiring over 5 days.
Purpose of the Study:
- To develop and validate species-specific polymerase chain reaction (PCR) and oligonucleotide array assays for detecting Enterobacter sakazakii.
- To target the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region for enhanced specificity.
- To compare the speed and reliability of the new methods against conventional techniques.
Main Methods:
- Sequencing of the ITS region from six E. sakazakii strains and BLAST analysis of GenBank data.
- Design of two pairs of specific PCR primers and 10 oligonucleotide probes targeting the ITS sequence.
- Testing the specificity and efficiency of the developed PCR and array assays against 88 bacterial strains, including E. sakazakii and non-E. sakazakii species.
Main Results:
- Both PCR and oligonucleotide array assays demonstrated high specificity, accurately detecting all E. sakazakii strains without cross-reactivity.
- The assays achieved a sensitivity of 1.3 CFU/100 g in infant formula when combined with selective enrichment.
- The entire detection process, including enrichment, took only 48 hours, a significant reduction compared to the 5-day conventional methods.
Conclusions:
- The developed species-specific PCR and oligonucleotide array assays are reliable and rapid tools for detecting Enterobacter sakazakii.
- These novel methods offer a significant time advantage over conventional techniques, enabling faster identification of this dangerous pathogen.
- The assays are suitable for ensuring the safety of products like infant formula, contributing to the prevention of neonatal meningitis.
Abstract:
Enterobacter sakazakii has been implicated in a several form of neonatal meningitis with a high mortality rate. In the present study, the species-specific PCR and oligonucleotide array assays were developed to detect the 16S-23S rDNA internal transcribed spacer (ITS) of E. sakazakii. Two pairs of specific PCR primers and 10 oligonucleotide probes were designed by sequencing the ITS of six strains of E. sakazakii and BLAST of GenBank. The specificity and efficiency of the PCR and oligonucleotide array methods were tested against a panel of numerous strains from 88 different bacterial strains. All of the E. sakazakii strains generated positive signal, and no cross-reaction was observed with non-E. sakazakii strains in the PCR and oligonucleotide array detections based on ITS sequences. Sensitivity of the detections is 1.3 CFU/100 g infant formula with the selective enrichment. Both of the PCR and oligonucleotide array procedures take only 48 h including the enrichment culture, whereas the conventional methods required at least 5 days. This study demonstrated that both of the pathogenic detections are time-saved and reliable.
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