Probes and methods for detection of Escheridia coli and antibiotic resistance
Inventors
Haake, David A. • Churchill, Bernard M. • Liao, Joseph C. • Suchard, Marc A. • Li, Yang • Mastali, Mitra
Assignees
US GOVERNMENT REPRESENTED BY DEAPRTMENT OF VETERANS AFFAIRS • US Department of Veterans Affairs • University of California San Diego UCSD
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Publication Number
US-7763426-B2
Publication Date
2010-07-27
Expiration Date
2025-11-01
Abstract
Described are probes and methods for detecting pathogens and antibiotic resistance of a specimen. The method comprises contacting the specimen with a growth medium; and lysing the specimen to release nucleic acid molecules from the specimen. The lysate of the specimen is contacted with a capture probe immobilized on a substrate, wherein the capture probe comprises an oligonucleotide that specifically hybridizes with a first target nucleic acid sequence region of ribosomal RNA. The lysate is in contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second target nucleic acid sequence region of ribosomal RNA. The presence or absence of labeled oligonucleotide complexed with the substrate is determined. Detection of labeled oligonucleotide complexed with the substrate is indicative of the presence of pathogen. Performing the method in the presence and absence of an antibiotic permits detection of antibiotic resistance.
Core Innovation
The invention provides probes and methods for rapid, species-specific detection of pathogens, particularly bacterial uropathogens, in specimens such as urine. It addresses the need for quicker diagnostic methods compared to traditional urine culture, which typically requires 1-2 days for pathogen identification. The invention utilizes oligonucleotide capture and detector probes that specifically hybridize to regions of bacterial ribosomal RNA (especially 16S rRNA) to detect pathogens directly in lysed specimens using an electrochemical sensor array.
The method involves lysing the specimen to release nucleic acid molecules, contacting the lysate with a capture probe immobilized on a substrate and a detector probe that comprises a detectably labeled oligonucleotide. The presence of the labeled detector probe complexed with the substrate indicates the presence of the pathogen. Additionally, by inoculating the specimen into growth medium with and without antibiotics and measuring the pathogen presence via hybridization-based detection, the method also allows rapid detection of antibiotic resistance or susceptibility.
A universal lysis buffer is provided that effectively lyses both gram-positive and gram-negative bacteria, enabling immediate assay without requiring bacterial purification, thus accelerating the process. The probes are typically short oligonucleotides that hybridize under highly stringent yet ambient temperature conditions, allowing for rapid assays compatible with point-of-care testing. The invention also discloses kits comprising such probes and electrochemical sensor arrays pre-functionalized with capture probes for practical application.
Claims Coverage
The claims include independent claims directed to pairs of oligonucleotide probes and methods for detecting E. coli and its antibiotic susceptibility, as well as assay kits incorporating these components. Key inventive features relate to probe design, hybridization conditions, specimen lysis methods, and detection approaches.
Short oligonucleotide probes specific for E. coli 16S rRNA
A pair of oligonucleotide probes (capture and detector), each 10-15 bases in length, that specifically hybridize under highly stringent conditions (20° C. to 39° C. in 1M phosphate buffer at pH ~7) to adjacent regions of E. coli 16S rRNA, comprising sequences substantially corresponding to SEQ ID NO: 28 (capture) and SEQ ID NO: 29 (detector).
Method for detecting E. coli presence using short probes
A method comprising contacting a lysate of the specimen with an immobilized capture probe specific for SEQ ID NO: 28 and contacting the lysate with a detectably labeled detector probe specific for SEQ ID NO: 29, both probes 10-15 bases long and sharing 100% complementarity to said sequences, under hybridization conditions (20° C. to 39° C. in buffered saline), and determining the presence of the detector probe bound to the substrate to indicate E. coli presence.
Specimen lysis method employing non-denaturing detergent and lysozyme, optionally combined with NaOH
Preparation of the lysate by contacting the specimen with a first lysis buffer comprising a non-denaturing detergent (e.g., Triton X-100) and lysozyme, optionally followed by or preceded by contact with a second lysis buffer comprising NaOH, at 20° C. to 39° C. for about 5 minutes each, facilitating nucleic acid release suitable for probe hybridization.
Detection of antibiotic susceptibility using nucleic acid hybridization
A method detecting antibiotic susceptibility comprising identifying and quantifying E. coli using the probe hybridization method, inoculating the specimen into growth medium with and without antibiotic, and comparing the amount of labeled oligonucleotide complexed with substrate in each condition, where reduced labeled oligonucleotide presence in the antibiotic condition indicates susceptibility.
Assay kit comprising electrochemical sensor array with immobilized short probes
A kit comprising an electrochemical sensor array with electrodes immobilized with capture probes, each 10-15 bases long and complementary to adjacent regions of bacterial ribosomal RNA (specifically E. coli sequences SEQ ID NOS: 28 and 29), and a corresponding detector probe labeled detectable marker, optimized for hybridization at ambient to body temperature in 1M phosphate buffer.
The claims broadly cover short oligonucleotide probe pairs specific for adjacent 16S rRNA regions of E. coli used in rapid, ambient-temperature hybridization-based detection methods, specimen lysis techniques using detergent, lysozyme and NaOH, methods for antibiotic susceptibility testing by hybridization signal comparison, and assay kits utilizing electrochemical sensor arrays functionalized with these probes.
Stated Advantages
Rapid species-specific detection of bacterial pathogens significantly reduces the time from specimen collection to identification compared to traditional culture methods.
The universal lysis buffer obviates the need for separate lysis methods for gram-positive and gram-negative bacteria, enabling immediate assay without purification steps.
Electrochemical sensors offer sensitivity, selectivity, portability, and lower cost suitable for point-of-care molecular diagnostics.
The method allows phenotypic detection of antibiotic resistance, applicable to a broad range of organisms and antibiotics regardless of specific resistance mechanisms.
Short oligonucleotide probes permit hybridization at ambient temperature without denaturing agents, simplifying assay logistics and facilitating automation.
Documented Applications
Rapid molecular detection and species-specific identification of urinary tract infection pathogens in clinical urine specimens.
Use of an electrochemical sensor array for detection of bacterial 16S rRNA in bodily fluids including blood, saliva, sputum, semen, prostatic secretions, cerebrospinal fluid, and urine.
Rapid detection of antibiotic susceptibility or resistance in bacterial pathogens by comparing nucleic acid hybridization signals in the presence and absence of antibiotics.
Point-of-care diagnostic testing for infections using integrated sensor arrays and probe kits for rapid pathogen identification and antibiotic susceptibility testing.
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