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Validation of a Real-Time PCR Assay for Fully Automated Detection of Bacillus cereus in Donor Human Milk
Gemma Aran1,2, Vanessa Pleguezuelos1,2, Margarita Blanco1
1Banc de Sang i Teixits (Blood and Tissue Bank of Catalonia), 08005 Barcelona, Spain.
Abstract:
Donor human milk (DHM) can harbor microbial contaminants that cause serious infections in premature infants. Bacillus cereus is a pathogen frequently found in DHM, capable of forming spores that can resist Holder pasteurization (62.5 °C, 30 min). Since no microbial growth is acceptable in post-pasteurized DHM, microbiological testing of pre-pasteurized DHM provides information about its contamination level to determine if it should be accepted for pasteurization. Culture is the gold standard in microbiological control but it requires 24-48 h to provide results. In this study we developed and validated a non-commercial real-time PCR assay for the detection of Bacillus cereus (BC test) in DHM specimens on a fully automated high-throughput platform, the cobas® 6800 system. The BC test showed excellent sensitivity and specificity, repeatability and linearity over an 8-log range and a low limit of detection in milk specimens, as well as good agreement with selective culture methods. BC test was then used to systematically control all milk donations (3439) over a 24-month period. Bacillus cereus was detected in 14.2% of DHM, with monthly rates ranging from 6 to 29% and a significantly higher incidence in warmer months. Incorporating this assay into our laboratory workflow improved efficiency and reduced turnaround time.
Insights
A new real-time PCR assay rapidly detects Bacillus cereus in donor human milk (DHM), ensuring infant safety. This method improves efficiency and reduces testing time for DHM microbiological control.
Area of Science:
- Microbiology
- Food Safety
- Clinical Diagnostics
Background:
- Donor human milk (DHM) can be contaminated with pathogens like Bacillus cereus, posing infection risks to premature infants.
- Bacillus cereus spores are heat-resistant, surviving standard pasteurization methods.
- Current culture-based methods for microbial detection are time-consuming, delaying DHM screening.
Purpose of the Study:
- To develop and validate a real-time PCR assay for rapid and accurate detection of Bacillus cereus in DHM.
- To assess the performance of the PCR assay compared to traditional culture methods.
- To implement the validated assay for routine microbiological control of DHM donations.
Main Methods:
- Development and validation of a non-commercial real-time PCR assay for Bacillus cereus detection.
- Utilized the cobas® 6800 system, a fully automated high-throughput platform.
- Compared PCR results with selective culture methods for agreement assessment.
Main Results:
- The real-time PCR assay demonstrated excellent sensitivity, specificity, repeatability, and linearity.
- The assay exhibited a low limit of detection in milk specimens.
- Bacillus cereus was detected in 14.2% of 3439 DHM donations over 24 months, with seasonal variations.
Conclusions:
- The validated real-time PCR assay is a reliable and efficient tool for Bacillus cereus detection in DHM.
- Implementation of the assay significantly reduced laboratory turnaround time and improved workflow efficiency.
- This rapid detection method enhances the safety of DHM for vulnerable infant populations.
