This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zucol, F. Articles by Nadal, D. Search for Related Content PubMed PubMed Citation Articles by Zucol, F. Articles by Nadal, D.

Real-Time Quantitative Broad-Range PCR Assay for Detection of the 16S rRNA Gene Followed by Sequencing for Species Identification

Division of Infectious Diseases,¹ Division of Oncology, University Children's Hospital of Zurich, Zurich,² Division of Oncology, University Children's Hospital of Bern,³ Institute of Infectious Diseases, University of Bern, Bern,⁴ Bio-Analytica AG, Lucerne, Switzerland⁵

Received 18 January 2006/ Returned for modification 6 May 2006/ Accepted 9 June 2006

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of 10² CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

This article has been cited by other articles:

• Lever, A., Mackenzie, I. (2007). Sepsis: definition, epidemiology, and diagnosis. BMJ 335: 879-883 [Full Text]  
• Pingle, M. R., Granger, K., Feinberg, P., Shatsky, R., Sterling, B., Rundell, M., Spitzer, E., Larone, D., Golightly, L., Barany, F. (2007). Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay. J. Clin. Microbiol. 45: 1927-1935 [Abstract] [Full Text]