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https://www.arca.fiocruz.br/handle/icict/43572
OPTIMIZED BROAD-RANGE REAL-TIME PCR-BASED METHOD FOR BACTERIAL SCREENING OF PLATELET CONCENTRATES
Real-Time Polymerase Chain Reaction
Molecular Diagnostic Techniques
Blood Platelets
Reacción en Cadena en Tiempo Real de la Polimerasa
Técnicas de Diagnóstico Molecular
Plaquetas
Reação em Cadeia da Polimerase em Tempo Real
Técnicas de Diagnóstico Molecular
Plaquetas
Affilliation
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
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.
Keywords
Bacterial InfectionsReal-Time Polymerase Chain Reaction
Molecular Diagnostic Techniques
Blood Platelets
Keywords in Spanish
Infecciones BacterianasReacción en Cadena en Tiempo Real de la Polimerasa
Técnicas de Diagnóstico Molecular
Plaquetas
DeCS
Infecções BacterianasReação em Cadeia da Polimerase em Tempo Real
Técnicas de Diagnóstico Molecular
Plaquetas
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