Transfusion-associated septic reactions (TAS), that occur due to transfusion of blood and blood components contaminated with bacteria are often fatal. Red blood cells(RBC), which are stored at 4 C, are most frequently contaminated with Gram negative bacteria such as Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens etc. Bacteria associated with platelet concentrates, which are stored at room temperature, most often are the skin associated Gram positive coccis, such as Staphylococcus epidermidis, Bacillus cereus and Staphylococcus aureus and Gram negative bacteria such as Enterobacter cloacae, Klebsiella spp and Serratia marcescens. Methods for rapid and sensitive detection of these bacteria are lacking, as is knowledge of the true incidence rate of bacterial contamination of blood products. The existing methods depend on a bacterial concentration of about 10(4) - 10(7) bacteria /ml, before the bacteria can be detected. Nucleic-acid - based testing for detection, such as the polymerase chain reaction (PCR) is extremely sensitive and has the theoretical potential of detecting one bacterium in a sample within hours. Moreover it can identify the species of contaminating bacteria. I have developed a highly sensitive PCR assay for detecting Y. enterocolitica in blood samples stored up to 42 days at 4 C. Y. enterocolitica accounts for 50% of the clinical sepsis caused by RBC contamination. To detect it, I have chosen the fluorogenic 5'- nuclease assay, (the TaqMan PCR assay), which has the following advantages. (1) It is a real time PCR assay, and uses a fluorogenic oligonucleotide probe in the reaction. Thus, it has the potential of detecting targets in less than 2 hours. (2) It is a highly quantitative PCR that can indicate how many copies of the gene are present in a unknown sample. (3) It easily lends itself to automation. I have designed primers and probe targeted to the 16S rRNA gene region of Y. enterocolitica. There are several copies of this gene per cell and during active cell growth the abundance of the rRNA can be as high as 10(4) copies per cell, meaning that fewer target bacteria are needed to detect a contaminated unit. Several kits for purifying chromosomal DNA from blood that has been spiked with bacteria have been evaluated; hemoglobin, which is a potent inhibitor of Taq polymerase must be efficiently removed, and extraction reagents must not interfere with the fluorescence. The sensitivity acheieved with the TaqMan PCR assay for Y. enterocolitica was 30 bacteria per ml of blood. The primers and probe are specific for Y. enterocolitica and did not detect any other Yersinia species. In the second phase of the project the Y. enterocolitica assay model has been used to detect a broader spectrum of contaminating bacteria. The species included are Yersinia, Serratia, Klebsiella and Enterobacter. The rRNA gene has conserved DNA regions interspersed with variable sequences, in related bacteria. Thus a """"""""universal"""""""" forward and reverse primer set directed towards the conserved regions shared by these bacteria species has been designed. Two probes, one specific for all Yersinia species and a second probe specific for all Serratia, Klebsiella and Enterobacter species reported to contaminate blood, have been used in a single multiplex TaqMan PCR assay. The DNA extraction method was modified slightly, and the sensitivity achieved was 12-16 bacteria per ml of whole blood. I am in the process of designing primers and probes from the 16S rRNA region of 17 bacteria that commonly contaminate platelets. Platelets will be seeded with these bacteria and the total DNA extracted from these samples to be used in a single multiplex TaqMan assay.
