Antibiotic susceptibility testing using probes for preribosomal RNA
Inventors
Haake, David A. • Halford, Colin • Babbitt, Jane T. • Churchill, Bernard M.
Assignees
US Department of Veterans Affairs • University of California San Diego UCSD
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Publication Number
US-10370728-B2
Publication Date
2019-08-06
Expiration Date
2033-05-03
Abstract
Described are probes and methods for detecting antibiotic susceptibility of a specimen. The method comprises contacting the specimen with an oligonucleotide probe that specifically hybridizes with a target nucleic acid sequence region of ribosomal RNA. The target sequence is at the junction between a pre-ribosomal RNA tail and mature ribosomal RNA of 23S or 16S rRNA. Performing the method in the presence and absence of an antibiotic permits detection of antibiotic susceptibility.
Core Innovation
The invention provides probes and methods for detecting antibiotic susceptibility of bacteria by contacting a specimen with an oligonucleotide probe that specifically hybridizes to a target nucleic acid sequence region of ribosomal RNA, specifically at the junction between pre-ribosomal RNA (prRNA) tail and mature ribosomal RNA (mrRNA) of 23S or 16S rRNA. By performing hybridization in the presence and absence of an antibiotic, the method detects antibiotic susceptibility by observing reduced probe hybridization when antibiotic is present.
The background identifies an urgent need for rapid and convenient methods to detect and test antibiotic susceptibility in clinical specimens, to guide diagnosis and treatment of infectious diseases. Treatment is often started before pathogen identification and antibiotic sensitivity are known, which hampers effective therapy. The invention addresses this need by providing a method that detects antibiotic susceptibility rapidly by measuring changes in pre-rRNA levels using specific probes.
In the summary, the method entails contacting bacterial samples with probes that specifically bind to rRNA sequences spanning the prRNA-mrRNA splice site. These probes may be single or pairs that hybridize contiguous sequences including the splice site. A significant reduction in probe hybridization in the presence of antibiotic compared to absence indicates susceptibility. The method may be conducted without pre-treatment to deplete prRNA, facilitating rapid processing. The invention also includes devices comprising such probes immobilized on solid supports, kits with the probes, and methods for monitoring bacterial growth rate using the pre-rRNA splice site as a marker.
Claims Coverage
The patent includes one independent device claim and three independent method claims, which cover the detection device and methods for determining antibiotic susceptibility, antibiotic efficacy, and bacterial growth rate using specific oligonucleotide probes.
Device comprising specific oligonucleotide probe pairs for intact pre-rRNA detection
A device comprises a pair of oligonucleotide probes, including sequences of SEQ ID NO: 5 and SEQ ID NO: 6, with at least one probe immobilized on a solid support. The probes selectively hybridize to a contiguous bacterial rRNA sequence spanning the splice site between prRNA tail and mature rRNA, enabling detection of intact pre-rRNA.
Method for determining bacterial susceptibility to antibiotics by comparative probe hybridization
A method involves contacting bacterial specimens with the device in the absence and presence of an antibiotic agent, detecting the relative amounts of probe hybridization, and identifying the sample as susceptible if the hybridization amount decreases by at least 80% in the presence of antibiotic.
Method for determining antibiotic efficacy of candidate agents using pre-rRNA probe hybridization
This method applies the device to specimens with and without a candidate antibiotic agent, detecting hybridization to target sequences spanning the pre-rRNA-mature rRNA splice site, and identifying the candidate agent as effective if hybridization is reduced by at least 80% in its presence.
Method for monitoring bacterial growth rate through changes in probe hybridization
A method for bacterial growth monitoring includes contacting a culture specimen with the device, detecting probe hybridization relative to an earlier time point, and identifying growth if hybridization increases, reflecting increased pre-rRNA levels during active growth.
The claims cover a device with specific oligonucleotide probes targeting the pre-rRNA-mature rRNA splice site and multiple methods using this device to detect intact pre-rRNA in bacterial samples to assess antibiotic susceptibility, antibiotic efficacy, and bacterial growth rate with high specificity and sensitivity.
Stated Advantages
The probes enable specific and selective detection of intact pre-rRNA at ambient temperature without requiring denaturing agents, facilitating rapid processing.
The method allows rapid determination of bacterial antibiotic susceptibility, improving therapeutic decision-making earlier than conventional methods.
The assay is sensitive enough to detect small changes in pre-rRNA levels, providing insights into bacterial growth phases and metabolic states.
Use of a ternary interface on gold electrodes maximizes hybridization efficiency and minimizes nonspecific background, enhancing assay sensitivity.
The device and methods can test novel antibiotic agents and guide treatment, including direct application to clinical specimens without requiring extensive sample preparation.
Documented Applications
Guiding diagnosis and treatment of subjects by determining antibiotic susceptibility in clinical specimens.
Testing efficacy of candidate antibiotic agents against bacterial samples using probe hybridization reduction.
Monitoring bacterial growth rate and physiological state in cultures by measuring relative amounts of pre-rRNA hybridization over time.
Detecting specific bacterial pathogens in bodily fluids and other samples via species-specific hybridization to pre-rRNA sequences.
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