Multiplex PCR for detection of Enterobacteriaceae in blood.
BACKGROUND
Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood.


METHODS
Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica.


RESULTS
When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection 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. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood.


CONCLUSIONS
The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.
Sen
K
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Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland, USA. Keya.Sen@uc.edu
Asher
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D001769:Blood / Q000382:microbiology*; D004755:Enterobacteriaceae / Q000302:isolation & purification*; D006801:Humans; D007711:Klebsiella pneumoniae / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012705:Serratia / Q000302:isolation & purification; D013997:Time Factors; D015008:Yersinia enterocolitica / Q000302:isolation & purification