The real-time fluorescence quantitative PCR assay for detection of bacterial contamination in platelet concentrates / 中国输血杂志

ABSTRACT

Objective To establish a real-time PCR assay to detect bacterial contamination in platelet concentrates(PCs).Methods PCs were spiked with serial dilutions of Staphylococcus aureaus and Eschetichia coli,and the bacterial genomic DNA was isolated by modified Chelex-100 method and then quantitatively detected by Tagman real-time PCR.Filtration was performed to avoid contamination from other bacterial DNA in the reagents of extraction kit as well as the PCR mixture.Results The real-time PCR targeting conservative 16S rRNA gene showed high sensitivity and specificity in detecting bacterial contamination in PCs,and no cross-reaction with human genomic DNA and viruses was found.The Ct value of the lowest bacterial dose groups after filtration in both S.aureaus and E.coli were statistically different from the negative controls,with a minimal detection quality of 0.3CFUs/PCR for S.aureaus,and 0.1CFUs/PCR for E.coli.,respectively.Conclusion The modified Chelex-100 method to extract bacterial genomic DNA and the subsequent real-time PCR are convenient,sensitive and specific,and may be applied to the large scale clinical detection of bacterial contamination in PCs.