Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Area of Science:

• Transfusion Medicine
• Microbiology
• Molecular Diagnostics

Background:

• Bacterial contamination of blood components, especially platelet concentrates (PCs), is a significant risk in transfusion medicine.
• Storage conditions for PCs can promote bacterial growth, increasing the risk of transfusion-transmitted infections.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and specific real-time PCR protocol for screening bacterial contamination in platelet concentrates.
• To improve the safety of transfused blood products by enabling timely detection of bacterial presence.

Main Methods:

• Development of an internally controlled real-time PCR assay targeting the bacterial 16S rRNA gene.
• Optimization using a proprietary 16S Universal PCR Master Mix and ethidium monoazide (EMA) to eliminate background DNA.
• Validation of the assay's sensitivity, specificity, and turnaround time.

Main Results:

• The real-time PCR protocol demonstrated high sensitivity, with a lower limit of detection at 10 genome equivalents (Ct value of 34±1.07).
• The method exhibited no non-specific amplification, ensuring specificity.
• The total processing and detection time, including DNA purification, was approximately 4 hours.

Conclusions:

• The developed real-time PCR method is a rapid, sensitive, and specific tool for screening bacterial contamination in platelet concentrates.
• This assay offers a significant reduction in detection time compared to traditional microbiological methods.
• Implementation of this method can enhance the safety of pre-transfusion platelet products.