A 16S ribosomal RNA TaqMan real-time reverse transcription PCR for specific detection of Salmonella in blood

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Abstract Background : The Gram -negative bacterium Salmonella enterica is an important human pathogen causing a huge public health burden worldwide. Reference diagnosis of Salmonella bloodstream infections (BSI) in patients is based on in vitro blood culture followed by biochemical and serotype identification. We have developed a TaqMan real-time reverse transcription PCR (RT-PCR) assay for the specific detection of Salmonella 16S rRNA molecules. The specificity was confirmed on a bacterial test panel grown in 5 ml BBL TM culture medium broth, comprising of six Salmonella enterica serovars (Typhi, Paratyphi A, Typhimurium, Enteritidis, Heidelberg and Weltevreden) and 10 non- Salmonella bacterial species that are known to cause human BSI.Results : The limit of detection (LOD) of the assay in serial dilutions of purified Salmonella enterica serovar Typhimurium RNA was 40 pg RNA per reaction, corresponding to ~2500 colony forming units (CFU). When applied directly in blood experimentally spiked with Salmonella Typhimurium, the assay showed an LOD of 10 5 CFU/ml which is 100x more sensitive than 16S rDNA detection using the same primers and probe set. In 10 ml whole blood spiked with Salmonella Typhimurium at 5 CFU/ml followed by incubation in an automatic blood culture instrument, the assay detected 16S rRNA after 10 hours incubation compared to 12 hours for 16S rDNA detection.Conclusion : Although prior in vitro incubation of blood samples is required, we here delivered the proof-of-concept of specific detection of Salmonella in clinical specimens by targeting its 16S rRNA molecule.

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