PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid

Abstract

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): BaciUus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, AUloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.

Molecular methods for pathogen and microbial community detection and characterization: Current and potential application in diagnostic microbiology

“…Nucleic acid sequencing of amplicon generated by broad-range PCR allows identification of the organism(s) by comparison with known sequences deposited at Genbank or other databases (Drancourt et al, 2000;Janda and Abbott, 2002;Petti, 2007). Previously uncharacterized microbes (novel pathogens) may also be detected because of the universal nature of primers/probes and the high sensitivity for detecting the presence of low copy number targets (Bottger, 1989;Relman and Falkow, 1992;Greisen et al, 1994;Monstein et al, 1996;Drancourt et al, 2000;Mothershed and Whitney, 2006;Procop, 2007).…”

“…The selection of primers and probes for broad-range PCR assays also effects analytical sensitivity. Primers targeting the 16S rRNA gene are used in most of these assays, while some have also targeted the 23S rRNA gene (Greisen et al, 1994). As outlined by Baker et al (2003), it is not possible to develop a 16S rRNA gene primer set that will be entirely universal.…”

“…As outlined by Baker et al (2003), it is not possible to develop a 16S rRNA gene primer set that will be entirely universal. Therefore, different conserved regions within the 16S rRNA gene and various primer sequences for been employed for analysis of human clinical specimens (Greisen et al, 1994;Baker et al, 2003). The target loci and the primer sequences impact taxonomic assignments.…”

New markers in pneumonia

“…Although these methods are highly specific, the result is affected by the usage of antimicrobial drugs and the sensitivity is poor. The application of polymerase chain reaction (PCR) in species-specific genes and conserved bacterial DNA sequence-based diagnostic tests has been developed [11,12]. In contrast to negative sputum cultures, most samples from patients infected with Streptococcus pneumoniae and receiving antibiotics can still be accurately evaluated by applying PCR [13].…”

Future Veterinary Diagnostics

“…Greisen and coworkers were one of the first groups to amplify fragments of the 16S rRNA gene using group-specific PCR primers and, with Southern blots that had pathogenspecific probes, identified pathogenic bacteria in cerebrospinal fluid. 32 Microarrays can be used in a similar fashion to interrogate PCR products to distinguish between multiple pathogens. A second PCR strategy is to use multiplex PCR to amplify a number of discreet, pathogen-specific genetic markers that are subsequently detected on a DNA microarray.…”