Electrochemiluminescent detection of bacteria in blood components.
Transfusion-transmitted bacterial infections cause significant patient morbidity and mortality. This study aimed to improve the sensitivity of a nucleic acid-based electrochemiluminescence (ECL) assay for pretransfusion bacterial testing of cellular blood components. The approach is dependent on the detection of bacterial 16S ribosomal RNA (rRNA). The modifications studied included the use of a chaotrope-based lysis buffer with high-energy mechanical cell disruption by RiboLysis, increased ruthenium (Ru2+) labelling per 16S rRNA molecule and concomitant use of fluorescent nucleic acid dyes (CyQUANT, Syto 17 red and Syto 61 red). The methodological changes made did lead to more effective bacterial cell disruption and enhanced ECL signal generation. Nevertheless, assay sensitivity was only slightly improved at approximately 10(4)-10(5) colony forming units per mL (CFU mL(-1)) and the results were highly inconsistent. The method is still not sensitive to the required 10(2) CFU mL(-1) and remains impractical for routine use in blood centres.
Rider
J
J
Bristol Institute for Transfusion Sciences, National Blood Service - Bristol Centre, Bristol, UK. janet.rider@nbs.nhs.uk
Newton
A
A
D016470:Bacteremia / Q000175:diagnosis; D001424:Bacterial Infections / Q000175:diagnosis* / Q000517:prevention & control / Q000635:transmission; D016913:Blood Component Transfusion / Q000009:adverse effects; D004563:Electrochemistry; D005456:Fluorescent Dyes; D006801:Humans; D008163:Luminescent Measurements; D008722:Methods; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000097:blood*; D012336:RNA, Ribosomal, 16S / Q000097:blood; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S