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

• Autor(a) principal: Alexandrino,F.
• Data de Publicação: 2021
• Outros Autores: Malgarin,J. S., Krieger,M. A., Morello,L. G.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Brazilian Journal of Biology
• Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842021000300692
• topic: bacterial contamination; real-time PCR; molecular testing; platelet concentrates

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.