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Multiplex PCR for detection of Enterobacteriaceae in blood
Author(s) -
Sen Keya,
Asher David M.
Publication year - 2001
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1046/j.1537-2995.2001.41111356.x
Subject(s) - enterobacter cloacae , microbiology and biotechnology , yersinia enterocolitica , biology , klebsiella oxytoca , serratia , enterobacteriaceae , klebsiella pneumoniae , enterobacter , taqman , bacteria , polymerase chain reaction , gene , escherichia coli , genetics , pseudomonas
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 Taq Man 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 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 Taq Man 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 Taq Man PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5′‐nuclease Taq Man PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.