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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 121,120 to Bergeron et al., entitled “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,” filed Apr. 11, 2002, which is a continuation of U.S. patent application Ser. No. 09 / 452,599, filed Dec. 1, 1999, now abandoned, which is a continuation of U.S. patent application Ser. No. 08 / 526,840, filed Sep. 11, 1995, now U.S. Pat. No. 6,001,564, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 304,732, filed Sep. 12, 1994, now abandoned.REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING [0002] The present application is being filed along with duplicate copies of a CD-ROM marked “Copy 1” and “Copy 2” containing a Sequence Listing in electronic format. The duplicate cop...

AI Technical Summary

Benefits of technology

"The present invention provides a method for detecting bacterial pathogens in clinical specimens using genomic DNA fragments specific to each bacterial species. These fragments can be derived from either bacterial genomic libraries or data banks. The use of oligonucleotide probes or primers allows for the rapid and accurate identification of bacterial species directly from clinical specimens, reducing the time required for bacterial detection and identification in the laboratory. The invention also provides a way to screen out negative clinical specimens and detect resistance genes in commonly encountered bacteria."

Problems solved by technology

Although the API and the microdilution systems are cost-effective, at least two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to isolate and identify the bacteria from the specimen.

However, this system has an unacceptable margin of error, especially with bacterial species other than Enterobacteriaceae (York et al., 1992.

Nevertheless, even this fastest method requires primary isolation of the bacteria as a pure culture, a process which takes at least 18 hours if there is a pure culture or 2 to 3 days if there is a mixed culture.

Although much faster, these rapid tests showed low sensitivities and specificities as well as a high number of false negative and false positive results (Koening et al., 1992.