Rapid detection of Cronobacter sakazakii by real-time PCR based on the cgcA gene and TaqMan probe with internal...

Area of Science:

• Food Microbiology
• Molecular Biology
• Public Health

Background:

• Cronobacter sakazakii is a highly virulent bacterium often found in powdered infant formula (PIF).
• Existing detection methods can be slow and lack specificity, posing a risk to infant health.
• A rapid and precise diagnostic tool is crucial for ensuring the safety of PIF.

Purpose of the Study:

• To develop and validate a fast and specific quantitative real-time PCR (qRT-PCR) assay for detecting Cronobacter sakazakii.
• To determine the assay's sensitivity, specificity, and detection limits in various matrices.
• To assess the assay's performance in the presence of competing microorganisms and potential inhibitors.

Main Methods:

• Development of a novel quantitative real-time PCR (qRT-PCR) assay targeting Cronobacter sakazakii.
• Validation of assay specificity using DNA from 46 bacterial strains, including 12 C. sakazakii strains and 34 non-target controls.
• Determination of detection limits in broth (CFU/mL) and PIF (CFU/g).
• Evaluation of the assay's performance after a 6-hour enrichment period in buffered peptone water.
• Testing the assay's ability to detect Cronobacter in PIF spiked with varying levels of C. sakazakii, in the presence of Lactobacillus pentosus and Bacillus cereus, and assessing its ability to handle inhibitors using an internal amplification control.

Main Results:

• The developed qRT-PCR assay demonstrated high specificity, correctly identifying all 12 C. sakazakii strains while showing no amplification in 34 non-target strains.
• The assay achieved a detection limit of approximately 110 CFU/mL in broth and 1100 CFU/g in PIF.
• After a 6-hour enrichment, the qRT-PCR could reliably detect as low as 2 CFU/25 g (0.08 CFU/g) of C. sakazakii in PIF.
• The assay successfully detected Cronobacter in spiked PIF samples even when co-inoculated with L. pentosus or B. cereus, which can inhibit bacterial growth and potentially lead to false negatives in culture-based methods.
• The internal amplification control confirmed the assay's robustness in detecting samples containing inhibitors.

Conclusions:

• The developed qRT-PCR assay is a rapid, specific, and sensitive method for detecting Cronobacter sakazakii in powdered infant formula.
• The assay's ability to detect low pathogen levels and function in the presence of inhibitors and competing microflora enhances its reliability for food safety applications.
• This qRT-PCR method offers a significant advancement for routine screening of PIF, contributing to improved infant health protection.