Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numb ers of bacteria that may sometimes contaminate units of blood during collec tion. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia e nterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probe s were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter speci es, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from p ure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneum oniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of det ection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y enterocolitica, S. liquefaciens, E cloac ae, or K pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.