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, are most often skin bacteria: Gram-positive cocci--such as Staphylococcus epidermidis and Staphylococcus aureus--Bacillus cereus, and Gram-negative bacteria such as Enterobacter cloacae, Klebsiella spp and Serratia marcescens. Methods to detect small numbers of these bacteria rapidly are lacking, as is knowledge of the true incidence rate of bacterial contamination of blood products. Existing methods require concentrations of about 10(4) to 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. Investigators in LBPUA 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.) The method used is a fluorogenic 5'-nuclease assay, (the TaqMan PCR assay), which has the following advantages: (1) It is a """"""""real-time"""""""" PCR assayusing a fluorogenic oligonucleotide probe in the reaction. Thus, it has the potential of detecting a target nucleic acid sequence in less than 2 hours. (2) It is a highly quantitative PCR that can indicate numbers of copies of a gene present in a unknown sample. (3) It easily lends itself to automation. LBPUA investigators designed primers and a 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 methods to purify chromosomal DNA from blood spiked with bacteria were 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 a second phase of the project the Y. enterocolitica assay model was used to detect a broader spectrum of contaminating bacteria: 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 was 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 were 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. In the next phase of the project, primers and probes will be selected from the 16S rRNA region of 17 bacteria that most 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. Progress in this project has been temporarily suspended following Dr. Sen's departure. Another investigator has been recruited to join LBPUA and to continue this research during the coming fiscal year.
