Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA-directed probes coupled to electrochemiluminescence.
Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1). Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively. This is equivalent to 10(5)-10(8)CFU mL(-1). These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.
Chaney
R
R
Bristol Institute for Transfusion Sciences, National Blood Service - Bristol Centre, UK.
Rider
J
J
Pamphilon
D
D
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D019921:Biotinylation; D001769:Blood / Q000382:microbiology*; D001792:Blood Platelets / Q000382:microbiology; D004566:Electrodes; D017707:Erythrocyte Transfusion; D004912:Erythrocytes / Q000382:microbiology; D006801:Humans; D008163:Luminescent Measurements; D008280:Magnetics; D009693:Nucleic Acid Hybridization; D017713:Platelet Transfusion; D011551:Pseudomonas fluorescens / Q000302:isolation & purification; D015347:RNA Probes; D012329:RNA, Bacterial / Q000097:blood*; D012335:RNA, Ribosomal / Q000097:blood*; D012428:Ruthenium; D013211:Staphylococcus aureus / Q000302:isolation & purification; D015008:Yersinia enterocolitica / Q000302:isolation & purification
D015347:RNA Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012428:Ruthenium