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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

a technology of amplification primers and probes, which is applied in the direction of bacteria peptides, biochemistry apparatus and processes, peptide sources, etc., can solve the problems of low sensitivities and specificities, system error margin, rapid test results, etc., to reduce rapid and sensitive bacterial identification, the effect of reducing the time currently required for identification

Inactive Publication Date: 2009-02-26
GENEOHM SCI CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]The development of hybridization (with either fragment or oligonucleotide probes) or of DNA amplification protocols for the detection of pathogens from clinical specimens renders possible a very rapid bacterial identification. This will greatly reduce the time currently required for the identification of pathogens in the clinical laboratory since these technologies can be applied for bacterial detection and identification directly from clinical specimens with minimum pretreatment of any biological specimens to release bacterial DNA. In addition to being 100% specific, probes and amplification primers allow identification of the bacterial species directly from clinical specimens or, alternatively, from an isolated colony. DNA amplification assays have the added advantages of being faster and more sensitive than hybridization assays, since they allow rapid and exponential in vitro replication of the target segment of DNA from the bacterial genome. Universal probes and amplification primers selected from the 16S or 23S rRNA genes highly conserved among bacteria, which permit the detection of any bacterial pathogens, will serve as a procedure to screen out the numerous negative clinical specimens received in diagnostic laboratories. The use of oligonucleotide probes or primers complementary to characterized bacterial genes encoding resistance to antibiotics to identify commonly encountered and clinically important resistance genes is also under the scope of this invention.

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.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0037]Isolation and cloning of fragments. Genomic DNAs from Escherichia coli strain ATCC 25922, Klebsiella pneumoniae strain CK2, Pseudomonas aeruginosa strain ATCC 27853, Proteus mirabilis strain ATCC 35657, Streptococcus pneumoniae strain ATCC 27336, Staphylococcus aureus strain ATCC 25923, Staphylococcus epidermidis strain ATCC 12228, Staphylococcus saprophyticus strain ATCC 15305, Haemophilus influenzae reference strain Rd and Moraxella catarrhalis strain ATCC 53879 were prepared using standard procedures. It is understood that the bacterial genomic DNA may have been isolated from strains other than the ones mentioned above. (For Enterococcus faecalis and Streptococcus pyogenes oligonucleotide sequences were derived exclusively from data banks). Each DNA was digested with a restriction enzyme which frequently cuts DNA such as Sau3AI. The resulting DNA fragments were ligated into a plasmid vector (pGEM3Zf) to create recombinant plasmids and transformed into competent E. coli cell...

example 2

[0040]Same as example 1 except that testing of the strains is by colony hybridization. The bacterial strains were inoculated onto a nylon membrane placed on nutrient agar. The membranes were incubated at 37° C. for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. DNA hybridization was performed as described earlier.

example 3

[0041]Same as example 1 except that bacteria were detected directly from clinical samples. Any biological samples were loaded directly onto a dot blot apparatus and cells were lysed in situ for bacterial detection. Blood samples should be heparizined in order to avoid coagulation interfering with their convenient loading on a dot blot apparatus.

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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.

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 a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled GENOM.046CP1CC5.TXT, created Aug. 20, ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N15/09C07K14/195C07K14/21C07K14/245C07K14/26C07K14/285C07K14/31C07K14/315C12N15/65C12Q1/06C12Q1/689C12R1/46
CPCC07K14/195C07K14/21C07K14/212C07K14/245C07K14/26C12Q1/689C07K14/31C07K14/315C07K14/3156C12N15/65C07K14/285C12Q2600/16
Inventor BERGERON, MICHEL G.OUELLETTE, MARCROY, PAUL H.
Owner GENEOHM SCI CANADA
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