Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories

Abstract

The present invention relates to DNA-based methods for universal bacterial detection, for specific detection of the common bacterial pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis as well as for specific detection of commonly encountered and clinically relevant bacterial antibiotic resistance genes directly from clinical specimens or, alternatively, from a bacterial colony. The above bacterial species can account for as much as 80% of bacterial pathogens isolated in routine microbiology laboratories. The core of this invention consists primarily of the DNA sequences from all species-specific genomic DNA fragments selected by hybridization from genomic libraries or, alternatively, selected from data banks as well as any oligonucleotide sequences derived from these sequences which can be used as probes or amplification primers for PCR or any other nucleic acid amplification methods. This invention also includes DNA sequences from the selected clinically relevant antibiotic resistance genes. With these methods, bacteria can be detected (universal primers and/or probes) and identified (species-specific primers and/or probes) directly from the clinical specimens or from an isolated bacterial colony. Bacteria are further evaluated for their putative susceptibility to antibiotics by resistance gene detection (antibiotic resistance gene specific primers and/or probes). Diagnostic kits for the detection of the presence, for the bacterial identification of the above-mentioned bacterial species and for the detection of antibiotic resistance genes are also claimed. These kits for the rapid (one hour or less) and accurate diagnosis of bacterial infections and antibiotic resistance will gradually replace conventional methods currently used in clinical microbiology laboratories for routine diagnosis. They should provide tools to clinicians to help prescribe promptly optimal treatments when necessary. Consequently, these tests should contribute to saving human lives, rationalizing treatment, reducing the development of antibiotic resistance and avoid unnecessary hospitalizations.

1 . A method to detect and identify the presence of  Staphylococcus aureus  and at least a second and a third target bacterial species in a sample by performing an assay, comprising:
 simultaneously contacting a sample with a set of amplification primers comprising a plurality of at least a first, second, and third primer pair, wherein said first primer pair hybridizes solely to the nucleic acids of  Staphylococcus aureus , and wherein said second and third primer pairs hybridize solely to target DNA of said second and third target bacterial species, respectively, and are ubiquitous to at least 80% to  Staphylococcus aureus , and second and third target bacterial species, respectively, wherein the plurality of primer pairs are chosen to allow amplification under a single amplification protocol;   amplifying target nucleic acid from said sample under said single amplification protocol; and   detecting the presence or amount of amplified product(s) as an indication of the presence of  Staphylococcus aureus  and said second and third target bacterial species in said sample.