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.
Highlights
Bacterial contamination of blood components is one of the major causes of transfusion-related infection
We developed a simple, rapid and sensitive broad-range real-time PCR protocol for bacterial screening of platelet concentrates (PCs) that eliminates the problem of co-amplification of contaminating microbial DNA
The PCR Master Mix was distributed in the 96-well plate (15 μL), wells were loaded with 5 μL of DNA template or water and real-time PCR was performed on a 7500 Real Time PCR System (Thermo Fisher Scientific)
Summary
Bacterial contamination of blood components is one of the major causes of transfusion-related infection. The BacT/ALERT system is used in Brazil for bacterial screening of PCs, which can detect 1-10 CFU per 5 mL within 24 - 48 hours (Albertoni et al, 2011) The sensitivity of such tests is directly proportional to the bacteria loads and the inoculated volume in a sample. We developed a simple, rapid and sensitive broad-range real-time PCR protocol for bacterial screening of PCs that eliminates the problem of co-amplification of contaminating microbial DNA. This protocol will be useful for assaying samples with low levels of contamination and to detect microorganisms that are difficult to grow in vitro or require a long period of incubation
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