# Validation of a Real-Time PCR Assay for Fully Automated Detection of Bacillus cereus in Donor Human Milk

## TL;DR

A new automated PCR test for detecting Bacillus cereus in donor human milk was developed and validated, improving detection efficiency and accuracy.

## Contribution

A novel non-commercial real-time PCR assay for Bacillus cereus detection in donor human milk was developed and validated on a fully automated platform.

## Key findings

- The BC test demonstrated excellent sensitivity, specificity, and linearity over an 8-log range in milk specimens.
- Bacillus cereus was detected in 14.2% of donor milk samples, with higher incidence in warmer months.
- The assay improved laboratory workflow efficiency and reduced turnaround time compared to traditional culture methods.

## 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.