Optimized broad-range real-time PCR-based method for bacterial screening of 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.
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Instituto Internacional de Ecologia
2021
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oai:scielo:S1519-698420210003006922021-03-29Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentratesAlexandrino,F.Malgarin,J. S.Krieger,M. A.Morello,L. G. 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.info:eu-repo/semantics/openAccessInstituto Internacional de EcologiaBrazilian Journal of Biology v.81 n.3 20212021-09-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842021000300692en10.1590/1519-6984.229893 |
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Alexandrino,F. Malgarin,J. S. Krieger,M. A. Morello,L. G. |
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Alexandrino,F. Malgarin,J. S. Krieger,M. A. Morello,L. G. Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
author_facet |
Alexandrino,F. Malgarin,J. S. Krieger,M. A. Morello,L. G. |
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Alexandrino,F. |
title |
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
title_short |
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
title_full |
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
title_fullStr |
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
title_full_unstemmed |
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates |
title_sort |
optimized broad-range real-time pcr-based method for bacterial screening of platelet concentrates |
description |
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. |
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Instituto Internacional de Ecologia |
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2021 |
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http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842021000300692 |
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